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Journal of Pharmaceutical Sciences xxx (2016) 1-23

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Journal of Pharmaceutical Sciences

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Review

Recent Advances in Lipid-Based Vesicles and Particulate Carriers forTopical and Transdermal Application

Shashank Jain 1, *, Niketkumar Patel 2, Mansi K. Shah 3, Pinak Khatri 4, Namrata Vora 5

1 Department of Product Development, G & W Labs, 101 Coolidge Street, South Plainfield, New Jersey 070802 Charles River Laboratories Contract Manufacturing PA, LLC, Boothwyn, Pennsylvania 190613 Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 775554 Department of Product Development, G & W PA Laboratories, Sellersville, Pennsylvania 189605 Department of Formulation Development, Capsugel Dosage Form Solutions Division, Xcelience, Tampa, Florida 33634

a r t i c l e i n f o

Article history:Received 17 June 2016Revised 2 October 2016Accepted 3 October 2016

Keywords:liposomesnanoparticlesdrug delivery systemstransdermal drug deliverypermeabilitypercutaneouscontrolled releasecolloidskinlipids

* Correspondence to: Shashank Jain (Telephone: þ1-5933).

E-mail address: shashank52@gmail.com (S. Jain).

http://dx.doi.org/10.1016/j.xphs.2016.10.0010022-3549/© 2016 American Pharmacists Association

a b s t r a c t

In the recent decade, skin delivery (topical and transdermal) has gained an unprecedented popularity,especially due to increased incidences of chronic skin diseases, demand for targeted and patientcompliant delivery, and interest in life cycle management strategies among pharmaceutical companies.Literature review of recent publications indicates that among various skin delivery systems, lipid-baseddelivery systems (vesicular carriers and lipid particulate systems) have been the most successful. Ve-sicular carriers consist of liposomes, ultradeformable liposomes, and ethosomes, while lipid particulatesystems consist of lipospheres, solid lipid nanoparticles, and nanostructured lipid carriers. These systemscan increase the skin drug transport by improving drug solubilization in the formulation, drug parti-tioning into the skin, and fluidizing skin lipids. Considering that lipid-based delivery systems areregarded as safe and efficient, they are proving to be an attractive delivery strategy for the pharma-ceutical as well as cosmeceutical drug substances. However, development of these delivery systemsrequires comprehensive understanding of physicochemical characteristics of drug and delivery carriers,formulation and process variables, mechanism of skin delivery, recent technological advancements,specific limitations, and regulatory considerations. Therefore, this review article encompasses recentresearch advances addressing the aforementioned issues.

© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Introduction

The pharmaceutical drug delivery market is expected to growfrom $1048.1 billion in 2015 to $1504.7 billion by 2020, with acompound annual growth rate of 7.5%.1 Conventionally and till todate, the oral route retains a major share of this drug deliverymarket. However, the oral route is becoming increasingly unpop-ular for variety of drugs and disease conditions, particularly due tothe recent technological advancements in drug delivery arena (suchas improvement in manufacturing processes, fabrication of func-tionalized polymers, and evaluation techniques) and emergingdemand for a more localized delivery to minimize side effects. Inthis regard, skin delivery provides an attractive alternative to oraldrug delivery.1 Skin delivery can be broadly differentiated into

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dermal (topical) and transdermal drug delivery. Dermal delivery isthe application of drug directly at the site of action (skin surface),resulting in higher localized drug concentration with reducedsystemic drug exposure.2 On the other hand, transdermal deliverytransports the drug across skin surface to the systemic circulationfor achieving therapeutic levels. Both topical and transdermal ap-plications have successfully delivered variety of drugs.1,3-5 This isalso evident from the fact that skin delivery which was valuedaround $9.44 billion in 2013 is expected to reach $11.21 billion by2018 with higher compound annual growth rate compared to theoral route. The main driving force for the increasing interest in theskin delivery could be attributed to the increasing incidences ofchronic skin diseases, demand for targeted and patient compliantdelivery, highly competitive oral drug deliverymarket, and growinginterest among pharmaceutical companies in life cycle manage-ment strategies.1,2,6

However, despite growing interest in the skin delivery, thegreatest challenge for the researchers is to overcome the inherentlimitation of drug absorption imposed by impervious stratum

hts reserved.

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-232

corneum, the outermost layer of the skin.6,7 Researchers have triedvarious approaches to either disrupt or weaken the stratum cor-neum to improve skin delivery. The first major approach to over-come the skin barrier is the use of chemical enhancers such asazones, glycols, ethanol, terpenes, and so on.6,8 They facilitate drugtransport by partially fluidizing skin lipids and increasing drugpartitioning. A second approach is to use physical enhancementmethods, such as sonophoresis (ultrasound), electroporation,magnetophoresis, microneedles, thermal ablation, micro-dermabrasion, and iontophoresis.7,9-12 This approach bypasses thestratum corneum and delivers the drug directly to the target skinlayer. Both of the aforementioned approaches have shown suc-cessful delivery for variety of drugs.11,13 However, physical ap-proaches are mostly painful, expensive, and lack patientcompliance while chemical permeation enhancers can cause skinirritation and permanent skin damage.6 Finally, the third approachis the use of drug delivery systems like nanoparticles, microparti-cles, and lipid-based delivery systems. These systems can increaseskin transportation by improving drug solubilization in theformulation, drug partitioning into the skin, and by fluidizing theskin lipids.6 Among the various studied drug delivery systems,lipid-based delivery systems have shown a great potential for bothtopical and transdermal delivery, especially in the last fewdecades.14

Lipid-based delivery systems are composed of biocompatibleand biodegradable lipids that can be utilized for controlledrelease, targeted delivery, and drug protection. The first com-mercial product utilizing lipid-based delivery system was mar-keted in 1988 for antimycotic agent, econazole.15 Since then,several reports are published indicating the success of these de-livery systems.6,14,16-19 Based on the recent literature review forskin application, majority of the lipid-based skin delivery systemsare classified into vesicular carriers and lipid particulate systems.Vesicular carriers comprise liposomes, ethosomes, ultra-deformable liposomes, and other specialized novel vesicular car-riers. Due to the limited success of conventional liposomes in theskin delivery, majority of the recent research are predominantlyfocused on polymeric liposomes (PLs) and elastic liposomes likeultradeformable liposomes and ethosomes. Lipid particulate sys-tems have also gained popularity in the recent past. Among thisclass, lipospheres, solid lipid nanoparticles (SLNs), and morerecently nanostructured lipid carriers (NLCs) have been success-fully utilized for skin delivery. Table 1 provides a brief summary ofvarious lipid-based delivery systems.

The lipid-based delivery systems can be tailored to targetvarious skin conditions depending on the delivery system selected,formulation composition, manufacturing processes, and processvariables. However, fabrication of these delivery systems requiresunderstanding of process and formulation variables, mechanism ofskin delivery, knowledge of physicochemical characteristics, recenttechnological advancements, and specific limitations. To addressthese needs, this review article focuses on lipid-based deliverysystems (specifically vesicular and lipid particulates) withemphasis on recent research, advancements, and challenges. Also,acknowledging that the literature provides only a limited review onlipid-based delivery systems for unique areas like transcutaneousimmunization (TCI), vaccine delivery via the skin, and cosmeceut-icals, we have attempted to encompass these areas within thelimited scope of this review article. Furthermore, it is also imper-ative to understand the associated regulatory implications forachieving commercial success of these delivery systems for skinapplication. However, because literature review of past decadeprovides little information of this subject, the regulatory aspectsand U.S. Food and Drug Administration (FDA) standpoint for lipid-based delivery systems are also covered in this article.

Skin Anatomy and Physiology

The skin is the largest organ of the human body. The total surfacearea of the skin of an averagemale adult is approximately 2m2.35 Themajor functions of the skin include protection against mechanicalstresses, preventionof excessivewater loss; facilitating transpirationalcooling, and preventing absorption of foreign bodies. Anatomically,skin is composed of 3 main distinguishable layers, namely epidermis,dermis, and subcutaneous (SC) “fat” tissues (Fig. 1).36

Epidermis

The epidermis is divided into 2 regions: the nonviable epidermis(the stratum corneum) and the viable epidermis. It consists of 70%water and keratinizing epithelial cells responsible for synthesis of thestratumcorneum.37 The epidermis does not contain anybloodvesselsand hencemolecules permeating across the epidermismust cross thedermal-epidermal layer to enter the body’s systemic circulation.

The stratum corneum is the outermost layer of the skin and isinvolved in skin homeostatic and protective functions. The stratumcorneum is the final product of epidermal differentiation withapproximately 10-20 mm thickness and is considered as metaboli-cally inactive.37 It consists of 10-25 layers of dead, elongated, fullykeratinized corneocytes, which are embedded in a matrix of thelipid bilayers. It typically resembles “Brick and Mortar” typestructure, where corneocyte from hydrated keratin of the skin re-sembles Bricks embedded in a Mortar, comprising of extracellularlipid components.38 The extracellular lipid is constituted of 2lamellar phases with predominant crystalline phase and the sub-population of liquid lipid phase.39 Lipids that constitute the extra-cellular matrix of the stratum corneum have a unique compositionand are very different from the lipids that constitute most biolog-ical membranes.

The viable epidermis is present below the stratum corneum and isapproximately 50-100 mm thick.40 It is different from the stratumcorneum because it is physiologically more closely akin to the otherliving cellular tissues and contains many metabolizing enzymes. Theviable epidermis is involved in the generation of stratumcorneumandmetabolismof the foreign substances. It is also involved in the immuneresponse of the skin due to the presence of Langerhan cells (LCs).41

Dermis

The dermis is a supportive, compressible, and elastic con-nective tissue protecting the epidermis. It is composed offibrous proteins (collagen and elastin) and an interfibrillar gel ofglycosaminoglycans, salts, and water. Blood and lymphaticvessels, nerve endings, hair follicle, sebaceous glands, and sweatglands are embedded within the dermis. Extensive vascularnetwork in the dermis plays a crucial role in skin nutrition,repair, immune responses, and thermal regulation.37 The hairfollicles and sweat ducts form a direct connecting path fromdermis to the skin surface, bypassing stratum corneum andhenceforth involved in providing appendageal route of skinpermeation.42

Subcutaneous “Fat” Tissue

The SC fat tissue located below the dermis is composed ofthe cells that contain large quantities of fat, making thecytoplasm lipoidal in character.37 The collagen between the fatcells provides the linkage of the epidermis and the dermiswith the underlying structures of the skin. The main functionof SC fat tissue is to act as a heat insulator and shockabsorber.

Table 1Summary of Lipid-Based Delivery Systems

Lipid-Based Delivery System Definition Typical FormulationComposition

Advantages Challenges

Vesicular carriersLiposomes20,21 These are conventional vesicles (single or multilayers)

that are formed when biodegradable lipids(phospholipid and cholesterol) come into contactwith the aqueous medium, wherein the hydrophilichead group of the lipid surrounds the aqueous corewhile the hydrophobic tail group is exposed to theexternal medium

PhospholipidCholesterolAqueous medium

Lipids are biocompatible and biodegradable High cost of lipids in general. Synthetic lipids are evenmore expensive than natural lipids

Well-studied manufacturing (conventional)processes and its process parameters atlaboratory scale

Process scalability challenges for commercialapplication along with risk of residual organicsolvent in the drug product

Suitable for both hydrophobic and hydrophilicdrug loading

Poor chemical (e.g., oxidative degradation) andphysical stability (e.g., aggregation and fusion)

Improves localized delivery Poor permeation to viable epidermis and dermisLack of well-established regulatory guidance for skin

deliveryPoor physicochemical characteristics (higher particle

size, higher rigidity, and low encapsulationefficiency)

Ultradeformable liposomes(also called astransferosomes ordeformable liposomes)22-24

These are elastic liposomes similar to conventionalliposomes in terms of its preparation techniquesand vesicular structure but functionally they aresufficiently deformed due to presence of edgeactivator

PhospholipidEdge activatorAqueous medium

Lipids are biocompatible and biodegradable High cost of lipidsManufacturing process and process parameters

are similar to that of liposomes (which areextensively studied at laboratory scale)

Process scalability challenges for commercialapplication along with risk of residual organicsolvent in the drug product

Higher elasticity and smaller vesicle size thanconventional liposomes due to the presenceof edge activator

Hydrophobic drug loading can compromise elasticityof these vesicles

Higher skin permeation potential compared toconventional liposomes

Limited skin permeation under occlusive condition

High membrane hydrophilicity and elasticityfacilitate these vesicles to avoid aggregationand fusion under osmotic stress, which posesa problem to the conventional liposomes

Lack of well-established regulatory guidance for skindelivery

Ethosomes25,26 These are elastic liposomes similar to conventionalliposomes in terms of its preparation techniquesand vesicular structure but functionally they aresufficiently deformed due to the presenceof ethanol

PhospholipidCholesterolWater and ethanol

cosolvent medium

Lipids are biocompatible and biodegradable High cost of lipidsManufacturing process and process parameters

are similar to that of liposomes (which areextensively studied at laboratory scale)

Process scalability challenges for commercialapplication along with risk of residual organicsolvent in the drug product

Suitable for both hydrophobic and hydrophilicdrug loading

Lack of long-term structural and chemical stabilitydata during storage

Higher elasticity, smaller vesicle size and higherentrapment efficiency than conventionalliposomes.

Challenge in optimizing lipid and ethanolconcentration to achieve improvedphysicochemical properties without compromisingstability of the ethosomes

Unlike ultradeformable liposomes, it enhancesskin permeation under both occlusive andnonocclusive conditions

Lack of well-established regulatory guidance for skindelivery.

Higher skin permeation than conventional andultradeformable liposomes (in most cases).

Possibility of skin irritation and toxicity due to highethanol content

Lipid particulate systemsLipospheres27-29 These are microspheres, composed of solid

hydrophobic lipid core stabilized by amonolayer of phospholipid embeddedon the surface

Fats (mainly solidtriglyceride)

Stabilizer (e.g.,phospholipid)

Aqueous medium

Biodegradable and biocompatible Poor skin permeation compared to lipid-basedvesicles, SLNs, and NLC.

Relatively cost effective compared to lipid-basedvesicular carriers

Lack of long-term physical stability data

Ease of preparation and scale-up Higher particle size than lipid-based vesicular carriers,SLN, and NLC.

Possibility for extended release of entrappeddrug

Poor drug loading for hydrophilic compounds

Improved stability for photo-labile drugsControlled particle sizeHigh dispersability in aqueous medium

Lack of well-established regulatory guidance for skindelivery

(continued on next page)

S.Jainet

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ofPharm

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1-233

Table

1(con

tinu

ed)

Lipid-Based

DeliverySy

stem

Defi

nition

Typical

Form

ulation

Com

position

Adva

ntage

sChallenge

s

Solid

lipid

nan

oparticles

30-32

Theseareco

lloidal

lipid

nan

oparticles

composed

ofbiod

egradab

lesolid

lipop

hilicmatrix(attheroom

temperature

andbo

dytemperature)in

whichthe

drugmoleculescanbe

inco

rporated

Solid

lipid

Surfactant

Aqu

eousmed

ium

Biodeg

radab

lean

dbioc

ompatible

Dueto

highwater

content,ithas

tobe

generally

inco

rporated

into

semisolid

carriers

likeointm

ent

andge

lRelativelyco

steffectiveco

mpared

tolip

id-based

vesicu

larcarriers

Lack

oflong-term

physical

stab

ility

data.

Potential

expulsionof

active

compou

ndsduringstorag

eMan

ufacturingprocesses

arereproduciblean

dscalab

leGelationan

dco

nsequ

entlyparticleag

glom

eration

Avo

iduse

oforga

nic

solven

tduringthe

man

ufacturingprocess

Lack

ofwell-established

regu

latory

guidan

ceforskin

delivery

Smallerparticlesize

than

lipospheres

Protectdrugfrom

chem

ical

deg

radation

Flex

ibility

ofmod

ulatingdrugrelease

Nan

ostructuredlip

idcarriers

33,34

Theseareco

lloidal

nan

oparticles

producedby

mixing

liquid

lipid

(oils

)withthesolid

lipid

inwhichthe

liquid

lipid

iseither

embe

dded

into

thesolid

matrix

orlocaliz

edat

thesu

rfaceof

solid

particles

Solid

lipids

Liqu

idlip

ids(oils

)Su

rfactant

Aqu

eoussolution

Biodeg

radab

lean

dbioc

ompatible

Lack

oflong-term

physical

stab

ility

data

Man

ufacturingprocesses

arereproduciblean

dscalab

leLack

ofwell-established

regu

latory

guidan

ceforskin

delivery

Higher

drugload

ingcapacityco

mpared

toSL

Ns

Smallerparticlesize

than

lipospheres

Avo

id/m

inim

izepoten

tial

expulsionof

active

compou

ndsduringstorag

eLo

wer

water

contentco

mpared

toSL

Ns

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-234

Pathways for Skin Penetration

In accordance to the above-discussed Brick and Mortar model,the process of percutaneous absorption can occur via 2 differentroutes: transepidermal (intercellular and intracellular) and trans-appendageal (hair follicles, sweat ducts, and sebaceous glands)pathways (Fig. 2).36

Transepidermal Pathway

Transepidermal pathway consists of intercellular and intracel-lular pathways. Intercellular pathway involves solute diffusionthrough the intercellular lipid domains via tortuous pathway (viacornified cells of stratum corneum, the viable epidermis, and thedermis).43 Tracer studies have provided evidences that intercellularlipids, and not the corneocyte proteins, are the main epidermalpermeability barrier.44 Intercellular pathway was initially rejectedas a dominant skin permeation mechanism due to its small volumeoccupancy.43 However, later the intercellular volume fraction wasfound to be much larger than originally estimated.45,46 Thesestudies suggest that intercellular pathway provided a major resis-tance for skin permeation.

Intracellular (transcellular) pathway involves permeationthrough the corneocytes followed by the intercellular lipids.Compounds permeating through this route utilize the imper-fections in the corneocytes that create openings comprised ofwater. This route is therefore believed to prefer hydrophiliccompounds for delivery. It is interesting to note that the intra-cellular pathway requires not only partitioning into and diffu-sion through corneocytes but also into and across theintercellular lipids.47

Transappendageal Pathway

In transappendageal pathway, the penetrant traverse thestratum corneum via a “shunt” pathway provided by the hairfollicles or sweat glands. In particular, hair follicles play a majorcontributor for this pathway due to higher follicular distribution.Although the available surface area for the follicular route isassumed to be limited to approximately 0.1% of total skin surfacearea, it has recently been suggested that follicular number,opening diameter, and follicular volume are important consid-erations to define the extend of delivery.42,48 Also, the hair fol-licles extend deep into the dermis with significant increase inthe actual surface area available for the penetration. Manystudies have indicated the relevance of this pathway in skinpermeation.49-51

Principle of Skin Permeation

Passive permeation is the most simplistic scenario for skinpermeation and is governed via Fick’s first law of diffusion,where the rate of transfer (dQ/dt) of a solute through a mem-brane with unit area A in one dimension (x) is directly propor-tional to the concentration gradient (dc/dx) across themembrane. The permeation flux (J) can be mathematicallydefined as follows52:

J ¼ dQdt*A

¼ Ddcdx

(1)

As indicated in the equation, the permeation flux is directlyproportional to the concentration gradient across the membrane.The diffusion coefficient (D) can further be represented byEquation 2:

Figure 1. Schematic representation of anatomical structure of the human skin. Adapted with permission from Erdo et al.36

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-23 5

D ¼ BT6phR

(2)

where B is the Boltzmann constant, T the temperature, h the vis-cosity of the solute medium, and R the radius of the solute.

As indicated in Figure 3, Equation 1 can be represented asfollows:

J ¼ DA ðC1 � C2Þ

h(3)

In this equation, C1 and C2 are the concentrations across themembrane while h is the thickness of the membrane. Based onFigure 3, the partition coefficient (K) can be defined as follows:

Figure 2. The pathways for percutaneous absorptio

K ¼ C1Cd

¼ C2Cr

(4)

where Cd and Cr represent the concentration in the donor and re-ceptor compartment. Considering the partition coefficient,Equation 3 can be represented as follows:

J ¼ DAKðCd � CrÞh

(5)

It can be inferred that the passive diffusion of drug is dependenton the concentration gradient, temperature, viscosity of the solutemedium or delivery system, and the particle size of drug moleculeor delivery system.

n. Adapted with permission from Erdo et al.36

Figure 3. Permeation of drug molecule from donor compartment to receptor compartment across concentration gradient.

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-236

Lipid-Based Delivery Systems

Vesicular Carriers

The vesicular carriers have traditionally been used for topicaland transdermal drug delivery. They are typically composed ofbiocompatible lipids and aqueous phase (water, buffer solutions,or cosolvents). Structurally, these lipids form concentric lamellaeentrapping the aqueous phase. Owing to the lipophilic nature ofthe lipids, these vesicles (with entrapped drug) can supposedlypartition into the skin layers and deliver the drug across stratumcorneum. Additionally, because the vesicles are typically in nano-size range, they can further enhance the skin delivery of drug-loaded vesicular carriers. In general, it is suggested that vesiclesize �600 nm do not penetrate the deeper layers of the skin andstay in/or on the stratum corneum, vesicles �300 nm can pene-trate more deeply, but vesicles �70 nm can deliver to both theviable epidermal and dermal layers.53 For improving skinpermeation potential, researchers have invented and modifiedvarious vesicular carriers with unique structural and functionalproperties in the last 4 decades.

The first-generation lipid-based vesicular carrier was called li-posomes. The first reported publication in this field was fromMezeiand Gulasekharam in 1980.54,55 However, the success of liposomaldelivery was mainly limited by its vesicular size (typically 200-800nm) and rigidity, which can impede skin permeation.6,53 In 1992,Cevc and Blume introduced the second-generation vesicular car-riers named Ultradeformable liposomes or Transfersomes®, whichpossess smaller vesicular size (typically <300 nm) and higherelasticity (typically 5-8 times higher compared to conventionalliposomes).56,57 In 2000, Touitou et al.58 developed third-generation vesicular carrier called ethosomes. Ethosomes areethanol-based nanosized elastic lipid vesicles. The improved skinpermeation of ethosomes is attributed to the unique physico-chemical properties, that is, smaller vesicular size (typically <300nm) and higher elasticity (typically 10-30 times higher than con-ventional liposomes), as well as permeation enhancement effect ofethanol.6,57 More recently, various modifications of these vesicularcarriers are also studied to provide specific structural or functionalattribute for skin delivery.

Each of these vesicles has its specific features, mechanism ofdrug delivery, advantages, and challenges. The following sectiondiscusses the vesicular carriers in detail.

LiposomesConventional liposomes are one of the most famous and

extensively studied lipid vesicles, which are typically composed ofphospholipids, cholesterol, and aqueous medium (water or buffersolution with varying pH). These vesicles are formed when natu-rally or synthetically occurring biodegradable lipids come intocontact with the aqueous medium, wherein the hydrophilic headgroup of the lipid surrounds the aqueous core while the hydro-phobic tail group is exposed to the external medium. Due to thisunique structural property, water-soluble drugs can be loaded inthe aqueous core while the water-insoluble drugs can be loaded inthe lipid bilayer.

Although both natural and synthetic phospholipids are avail-able, conventional choice is often limited to naturally occurringphosphatidylcholines (e.g., soy or egg source) due to toxicologicalconsiderations and relative cost.59 Phosphatidylcholine is themajorcomponent of the liposomes and act as a permeation enhancer forskin delivery of the drugs. Due to the lower gel-liquid crystallinephase transition temperature, these lipids are in fluid state at theskin temperature of 32�C.6 The fluid-state phospholipids disturbthe rigid bilayer structure of the skin lipids leading to increase indrug partitioning into the lipid phase. Cholesterol is generallyadded to impart rigidity and stabilization by increasing the gel(stable) to liquid crystalline state (metastable) transition temper-ature of the lipid bilayer.60 However, the inclusion of cholesterol inthe liposome may decrease the encapsulation efficiency of hydro-philic drugs by reducing the volume of the aqueous phase.61

Furthermore, because the addition of cholesterol increases the ri-gidity of the vesicles, it can negatively impact the permeation ofthese vesicles through the skin.6

The most commonly used conventional techniques for liposomepreparation include thin-film hydration,6 reversed phase evapora-tion,62 and solvent injection techniques.63 Based on the availableliterature, among the aforementioned conventional techniques,thin-film hydration is most commonly used for skin deliverystudies. In this technique, lipids (phospholipid and cholesterol) are

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-23 7

dispersed in the organic solvent. Then, organic solvent is removedby means of evaporation (using a rotary evaporator at reducedpressure) leaving behind a dry lipid film on the wall of the flask.Finally, the dry lipid film is hydrated by aqueous phase (whilevortexing the content) to obtain liposomes. In this technique,processing parameters like hydration time, hydration temperature(temperature at which the lipids are hydrated by aqueous me-dium), and vortexing speed may affect various parameters (espe-cially vesicle size and entrapment efficiency) of the liposome andsubsequently may modify its skin permeation.64 For example, astudy conducted on liposomes prepared with imiquimod:phos-phatidylcholine:cholesterol weight ratio of 1:10:1 indicated thatincrease in hydration time from 90 to 150 min resulted in increasein entrapment efficiency from 36.85% to 65.32%, respectively.64

Also, hydration temperature higher than lipid phase transitiontemperature is preferred for this technique.6 However, most of theabove-mentioned conventional technologies encounter severedrawbacks. For example, thin-film hydration utilizes organic sol-vent and renders larger vesicle size liposomes.6 In case of solventinjection technique, a relatively dilute preparation of liposomes isobtained which decreases the encapsulation efficiency of theaqueous phase. Furthermore, most of these techniques exhibitscale-up issues. For detailed discussion on these conventionaltechniques and their challenges, the readers can refer to therecently published review articles.20,65

Recently, more advanced technologies such as supercriticalfluid,66,67 dual asymmetric centrifugation,68 and microfluidicchannels69,70 have been employed in liposome preparation for skindelivery application. Supercritical fluid technology provides agreen, nontoxic, inexpensive, and scalable alternative to the con-ventional liposome preparation techniques.71 Briefly, phospholipidand cholesterol are dissolved in supercritical CO2 and then allowedto precipitate in the form of ultrafine lipid particles. Afterwards,aqueous medium is added to consequently form liposome vesicles.Processing parameters like operational pressure, vessel tempera-ture, and flow rate ratio between CO2 and ethanol can affect variousproperties of the liposomes (especially drug loading, entrapmentefficiency, and particle size). In a recent study, effect of processparameters involved in supercritical fluid technology was studiedon CoQ10-loaded liposomes (phosphatidylcholine to drug weightratio of 10:1).66 It was observed that with decrease in the opera-tional pressure from 16 to 8 MPa, drug loading could increase up to4 times (2.95% and 8.92%, respectively), at constant vessel tem-perature of 35�C. Additionally, increase in temperature from 35�Cto 55�C can further improve drug loading from 8.92% to 10.2%,respectively (keeping all other parameters constant). Several re-searchers have shown promising results using supercritical fluidtechnology.72,73 Dual asymmetric centrifugation is another latesttechnology for liposome preparation.74 This is a unique advancedcentrifugation technique wherein 2 types of rotational forces areapplied. Conventional centrifugation rotational force moves thesample outward, while additional rotational force is provided tomove the sample toward the center of the centrifuge. This uniquecombination of 2 contra-rotational movements causes shearing ofthe sample (typically a dispersion of phospholipid, cholesterol, andaqueous medium) and consequently results in formation of the li-posomes. For model compound calcein, dual asymmetric centri-fugation technique was used on the concentrated blend ofhydrogenated phosphatidylcholine and cholesterol (55:45 mol%)and 0.9% NaCl solution. After optimization of process parameterslike centrifugation speed and time, the formed liposomes exhibitedparticle size of 60 ± 5 nm and entrapment efficacy of 56 ± 3.3%.74 Inanother study, siRNA (short-interfering RNAs) liposome, composedof phosphatidylcholine and cholesterol, was prepared using dualasymmetric centrifugation. The obtained liposomes resulted in

mean particle sizes of 79-109 nm with entrapment efficiencyranging from 43% to 81%. Additionally, based on spectral fluorim-etry, it was concluded that all entrapped siRNA was structurallyintact with no chemical degradation. Based on these results, thistechnology can be effectively utilized to load RNA (without causingdegradation problems) for skin delivery application.68 Anotherrecent but widely used technique is microfluidic channels whereliposomes are formed by passing the stream of alcoholic solution oflipid through 2 aqueous streams in a microfluidic channel.70,75-77

The laminar flow in the channels enables to control the size andsize distribution of the liposomes. It was demonstrated that lipo-some (cholesterol, dimyristoylphosphatidylcholine, and dihex-adecyl phosphate) vesicle size could bemodified from 50 to 150 nmby adjusting alcohol-to-aqueous volumetric flow rate.75

Various studies have shown the effect of formulation variables(e.g., lipid composition, type of lipid, drug-lipid ratio, concentrationand type of surface charge imparting compound, etc.) on thephysicochemical properties and skin permeation behavior of theliposomes.78-80 In our earlier work, we investigated the effect oflipid composition (phosphatidylcholine to cholesterol ratio) on thevesicle size, entrapment efficiency, elasticity, and skin permeationof diclofenac-loaded liposomes.6 It was observed that with increasein the phosphatidylcholine to cholesterol ratio from 50:50 to 90:10wt/wt, the vesicle size decreased (252-182 nm, respectively),entrapment efficiency increased (34.6%-53.6%, respectively), elas-ticity index increased (0.05-0.62, respectively), and in vitro cumu-lative drug permeate increased (0e94 mg/cm2, respectively). Theseresults were attributed to the presence of cholesterol that embedsinto the bilayer structure of the phosphatidylcholine, resulting inincrease in thickness (vesicle size), decrease in motion of the lipidtails (decreases elasticity), reduction in free volume for drugentrapment, and consequently decrease in drug permeationthrough skin. The type of lipid selected for liposome preparationalso needs to be carefully evaluated. For example, because egg-based phosphatidylcholine is more saturated than soy-basedphosphatidylcholine, liposomes prepared using the latter mayprovide a better oxidative stability.81 In another study, skinpermeation behavior of natural lipid (soy phosphatidylcholine andegg phosphatidylcholine) and synthetic lipid (hydrogenated soyphosphatidylcholine) was compared by preparing curcumin-loaded liposomes with phosphatidylcholine to cholesterol ratio of8:1.82 It was observed that although the particle size, entrapmentefficiency, and zeta potential were similar, the skin permeationbehavior was significantly different for natural and syntheticphospholipid-based liposomal formulations. Natural lipid-basedliposomal formulations exhibited higher skin permeation(approximately 1.5 times) and skin retention (approximately 1.7times) compared to synthetic lipid-based liposomal formulations.This behavior was attributed to the low phase transition temper-ature of the natural lipids (below 0�C), which results in increasedfluidity of the liposome and consequently enhances skinpermeation.

Another factor that can play a critical role is the surface charge ofthe lipid. Positively charged liposome has shown enhanced skinpermeation compared to neutral and negatively charged liposome,presumably due to interaction with negatively charged skinmembrane.19,83,84 More recently, drug-loaded liposomes are con-jugatedwith cationic cell penetrating peptide (CPP) to improve skinmembrane penetration of the liposomes.85 In a study, Polygonumaviculare L. extract (antioxidative and cellular membrane protectiveactivity) was loaded into CPP conjugated liposome for transdermaldelivery. In vivo studies indicated that the CPP conjugated lipo-somes were more effective in depigmentation and antiwrinklepotential than the conventional liposomes (without CPP). Thisresult was attributed to the ability of cationic peptide conjugated

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liposomes to effectively interact with the intercellular lipidlamellae of the stratum corneum compared to the conventionalliposomes.85 Similarly, for topical delivery of lidocaine (LID),transactivation transcriptional activator (TAT), one of the CPP wasconjugated on the octadecyl-quaternized lysine-modified chitosanPLs (TAT-PLs).86 The in vitro skin permeation results indicatedapproximately 4.17 and 1.75 times higher permeation flux ofLID-loaded TAT-PLs than that of LID solution and LID-loaded con-ventional liposome (composed of phosphatidylcholine andcholesterol), respectively. The author attributed the cationic PL(octadecyl-quaternized lysine-modified chitosan) and the posi-tively charged arginine group in TAT peptide sequences to facilitatebinding to the negatively charged skin membrane.86

Several theories have been proposed with regard to the mech-anism of skin delivery via liposomes. Some of the prominent the-ories include intact vesicular skin penetration,54,55 adsorptioneffect,87 and the penetration of liposomes through the trans-appendageal route.88,89 However, some researchers have recentlysuggested that the permeation enhancement effect of the liposomeis due to the interaction of liposome with the skin lipid causingpartial fluidization of skin lipid and consequently delivering thedrug to the deeper skin layers (below the stratum corneumlayer).87,90-92 Confocal laser scanning microscopy (CLSM) hasrevealed that conventional liposome might disintegrate and fusewith stratum corneum lipids, and consequently form a depot of thedrug on the skin surface.93 Thereafter, the extent of delivery will beguided by the physicochemical properties (solubility and partitioncoefficient) of the drug. This is evident from the limited success thathas been achieved in the field of liposomal skin delivery.94,95

Conventional liposomes are generally reported to be confined (ordisintegrates) in the upper layer of the stratum corneum andaccumulate in the skin appendages with minimal penetration tothe deeper skin layers, owing to their large size and lack ofelasticity.96

Literature review of past decade suggests that most of theliposome research is in the area of topical drug delivery. Liposomeshave been utilized for topical delivery of variety of drugs includingcurcumin,82,97 siRNA,98 loperamide,99 clotrimazole,100 resvera-trol,101 LID,102 and so on. Solubilizing ability of the liposomes wasutilized to load curcumin, a poorly water-soluble drug.82,97 It wasfound that liposomal curcumin with entrapment efficiency up to98% was 2-fold to 6-fold more potent than corresponding curcu-minoids. In another study, liposomes were utilized to deliver siRNAthrough skin for melanoma treatment.98 It was observed that li-posomes were able to not only penetrate into the skin layers butwere also effectively internalized into the viable cells of basalepidermis and knock down the target protein expression.98 Topicalapplication of loperamide hydrochloride-encapsulated liposomalgel (composed of phosphatidylcholine, cholesterol, and Carbopol®

940) resulted in potent and prolonged analgesic and anti-inflammatory activity compared to controls (free loperamide geland empty liposomal gel) in a rodent model.99

Despite various reported research work on conventional lipo-somes and PLs, clinical and commercial success of these vesicles arerather limited. This is due to the fact that skin permeation of theliposomes is mainly limited by its large vesicle size and lack ofelasticity.6 Furthermore, scalability of the manufacturing process,chemical instability, residual organic solvent in the drug product,liposomes aggregation, cost of lipids, and regulatory implicationsalso pose additional challenges in liposomes' success (Table 1).103

Ultradeformable LiposomesTo overcome some of the drawbacks of conventional liposomes,

a novel highly deformable elastic liposomes called ultradeformableliposomes (also called as Transferosomes® or deformable

liposomes) were introduced with the ability to penetrate the intactskin if applied nonocclusively.104 These elastic liposomes aresimilar to conventional liposomes in terms of its preparationtechniques and vesicular structure but functionally they are suffi-ciently deformed to penetrate pores (i.e., skin pores) much smallerthan their own size. Additionally, in contrast to the conventionalliposomes, the ultradeformable liposomes are made up of phos-pholipids, aqueous medium, and edge activators (Table 1). The edgeactivators are capable of increasing the deformability of the bilayerby affecting the interfacial tension of these vesicles. Transmissionelectron microscopy has conclusively demonstrated the deforma-tion of the vesicles into oval and irregular structures upon additionof edge activator.57,105,106 Another major difference between theultradeformable liposomes and the conventional liposomes is thehigher hydrophilicity of the former, which allows the elasticmembrane to swell more in comparison to the conventional lipidbilayer. High membrane hydrophilicity and elastic nature facilitatethese vesicles to avoid aggregation and fusion under osmotic stress,which poses a problem to the conventional liposomes.107

Apart from the formulation variables discussed in case of lipo-somes (e.g., type and concentration of lipid), the type and concen-tration of edge activator can significantly affect the physicochemicalproperties of these vesicles. Edge activators typically used forultradeformable liposome preparation include sodium cholate, so-diumdeoxycholate, Span 60, Span 65, Span 80, Tween20, Tween 60,Tween 80, and dipotassium glycyrrhizinate.108,109 In a study, theeffect of type of edge activators (sodium cholate, sodium deoxy-cholate, and Tween 80) on physicochemical properties of ultra-deformable liposomes (phosphatidylcholine to edge activatorweight ratio of 6:1) was evaluated. It was observed that sodiumcholate and sodiumdeoxycholate resulted in the smaller vesicle sizeand higher zeta potential compared to Tween 80.108 Ultra-deformable liposomes prepared with 95%:5% (wt/wt) (Phosphati-dylcholine:Edge activator) ratio showed entrapment efficiency inthe following orderdSpan 85 > Span 80 > Na cholate > Na deoxy-cholate > Tween 80dand it was attributed to the hydrophylliclipophyllic balance values of the respective edge activator.110 Inanother study, the effect of Tween 20 was studied on vesicularelasticityand the electron spin resonance study revealed that Tween20 increased the fluidity at the C5 atom of the acyl chain of thephospholipid (egg-based phophatidylcholine) bilayer.111

In addition, the concentration of edge activator plays a criticalrole as well. Ultradeformable liposomes (Lipid:Phospholipon 100®)prepared at different molar fractions of sodium cholate revealedthat increase in the molar fraction of sodium cholate >0.2 maycause formation of phospholipid/sodium cholate aggregates, suchas mixed vesicles, opened vesicles, mixed micelles, and rod-likemixed micelles, which can consequently lead to lower entrap-ment efficiency of the drug.112 Similarly, for diclofenac-loadedultradeformable liposomes (composed of phosphatidylcholineand Span 80), increase in concentration of Span 80 (edge activator)from 2% to 5% (wt/wt) resulted in increase in the entrapment effi-ciency from 50.73% to 55.19%, respectively.110 However, withfurther increase in edge activator concentration to 15% then 25%(wt/wt), the entrapment efficiency decreased from 44.93% to42.80%, respectively. The decrease in entrapment efficiency athigher concentration of edge activator was attributed to the for-mation of micelle aggregates.110

There are 2 major proposed mechanisms of skin delivery viaultradeformable liposomes. First mechanism proposes that intactvesicles enter the stratum corneum carrying drug molecules intothe skin.56 It is suggested that owing to the deformable nature,these vesicles are able to squeeze through the stratum corneum tothe deeper skin layers intact, under the influence of the naturallyoccurring transcutaneous hydration gradient. The skin surface is

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-23 9

relatively dry compared to the viable epidermis. When ultra-deformable liposomes are applied on the skin surface that ispartially dehydrated, the vesicles move toward the deeper skinlayers (e.g., viable epidermis and dermis) that are relatively hy-drated. The stress induced during the movement to deeper skinlayers is alleviated by the deformable nature of these vesicles. In anin vitro skin (200-300 mm thickness) permeation study, the pre-treatment of empty deformable liposomes on the skin surface fol-lowed by application of saturated aqueous solution of the drugs(pergolide or rotigotine) was compared with drug-loadeddeformable liposomes. It was observed that skin permeation wassignificantly higher in case of deformable liposome encapsulateddrugs compared to pretreatment of empty deformable liposomesfollowed by application of drug solution. This study suggests thatultradeformable liposomes may also act as carrier systems (ratherthan acting as a permeation enhancer for free drug) to deliver druginto deeper layers of the skin (up to 200-300 mmdeep from the skinsurface).113,114 However, researchers have reported that the hydra-tion gradient in the skin layer might not be linear. The water con-tent in the deeper region of the stratum corneum close to the viableepidermis is much lower than in the central regions of the stratumcorneum. Therefore, if hydration gradient is the driving force forultradeformable liposome delivery, it might be difficult for thevesicles to penetrate beyond the lowest layers of the stratum cor-neum.22 In another interesting study, CLSM indicated that fluo-rescein sodium-loaded ultradeformable liposomes utilizedtransfollicular pathway to penetrate to viable epidermis anddermis; however, the fluorescence intensity still remained higherin the stratum corneum region.115

Secondmechanism proposes that vesicles act as the penetrationenhancer, whereby vesicles enter the stratum corneum layers andsubsequently modify the intercellular skin lipids.71 This will facil-itate penetration of free drug molecules into and across the stratumcorneum. In a recent study, it was observed that deformable vesi-cles actually reduced the transdermal absorption of calcein, mostprobably by controlling the drug release from the formulation onthe skin surface.116 In an interesting study, skin permeation andskin deposition of ketotifen fumarate-loaded deformable liposomes(phosphatidylcholine to Tween 80 ratio of 84.5:15.5 wt/wt) andconventional liposomes (without Tween 80) were studied,respectively.117 It was observed that for deformable liposomes, skindeposition was 5 times higher than the skin permeation. Addi-tionally, even though skin deposition for deformable liposome wassimilar to conventional liposome, skin permeation of deformableliposome was significantly higher (2 times) than the conventionalliposomes. Based on these findings, it was suggested that deform-able liposome acts as penetration enhancer for the drug by inter-acting with the skin lipid.117 Despite various scientific effortssummarized above, it is still controversial whether the ultra-deformable liposomes act as a drug carrier or permeation enhanceror both.

Although the mechanism of skin delivery via deformable lipo-some is still unclear, researchers have successfully utilized ultra-deformable liposomes to deliver various drugs.57,106,112,118-120

Deformable liposomes composed of quercetin, phosphatidylcho-line, cholesterol, and Tween 80 showed 3.8-fold higher penetrationrate compared to it quercetin suspension.119 Similarly after 1 h ofnonocclusive incubation, the total accumulation of amphotericin inthe human skinwas 40 times higherwhen applied as amphotericin-loaded ultradeformable liposomes than as AmBisom (marketedamphotericin-loaded liposome).106 Itraconazole-loaded deform-able liposomes in the presence of hydroxypropyl-b-cyclodextrin(HP-b-CD) exhibited improvement in itraconazole delivery in stra-tum corneum and deeper skin layers compared to conventionalliposomes.105 In an attempt to find an alternative to the painful

penile injections for erectile dysfunction, topical deformable lipo-somes for papaverine hydrochloride (a vasoconstrictor)was studiedin 9 patients. Compared to control, statistically significantimprovement on pharmacodynamic responses were observed inthese patients.121 Ultradeformable liposomes have also been usedfor delivery of macromolecules via skin.112,122 The optimizedtransferosomal gel containing insulin showed good permeationbehavior with in vitro permeation flux of 13.50 ± 0.22 mg/cm2/hthrough porcine ear skin and demonstrated prolonged hypoglyce-mic effect in diabetic rats over 24 h.122 In another study, ultra-deformable liposomes provided a 10-fold increase in in vitro skinpermeation of asiaticoside compared to the free drug solution andfacilitated an increase in in vivo collagen biosynthesis.112

However, despite the success of ultradeformable liposomes,these vesicles possess some practical difficulties (Table 1). Becausetheir transport across the skin is driven by the hydration gradient,occlusive application can compromise the action of the deformablevesicles by eliminating the gradient force. Another major disad-vantage of these vesicles corresponds to the difficulty in loadinghydrophobic drugs into the vesicles without compromising theirdeformability and elastic properties.22

EthosomesEthosomes are new generation elastic lipid carriers; those have

shown enhanced skin delivery for both hydrophilic and lipophilicdrugs. Although ethosomes are conceptually sophisticated, thesimplicity involved in their preparation along with improved safetyand efficacy have made these vesicles suitable for skin delivery.25

The vesicular structure is composed mainly of phosphatidylcho-line, cholesterol, ethanol, and water. Preparation techniques arealso similar to conventional liposomes. Despite utilization oforganic solvent, thin-film hydration technique is generallypreferred for ethosomes because of its simplicity and highentrapment efficiency.123 Furthermore, unlike ultradeformable li-posomes, ethosomes can provide effective delivery under bothocclusive and nonocclusive conditions. Effectiveness of these ves-icles in skin delivery is attributed to its soft and elastic nature,instigated due to the presence of ethanol. Owing to their elasticnature, ethosomes are able to penetrate through the small poresand channels of the skin.58

In order to understand the elastic nature of ethosomes, it isimportant to understand the interplay of phosphatidylcholine,ethanol, and cholesterol on phase transition temperature (TP) of theethosome vesicles. Phosphatidylcholine has a characteristic TP, thatis, the temperature at which its gel state transitions into the liquidcrystalline state. In gel state, the molecular motion of the lipids isseverely restricted while in the liquid crystalline state the confor-mational disorder predominates resulting in smaller sized vesicleswith high elasticity.6 Cholesterol increases the TP value of the lipidvesicles making these vesicles more rigid and consequently morestable. On the other hand, ethanol interacts with the hydrophilichead group region of the phosphatidylcholine and eventually de-creases the TP of the lipid vesicles, facilitating the transition fromgel state into the liquid crystalline state. In our earlier investigation,we studied the effect of phosphatidylcholine (soy based) tocholesterol ratio (PC:CH) and ethanol on TP of the lipid vesicles. Itwas observed that with increase in cholesterol concentration from10% to 50% wt/wt, the TP value increased from 8.78�C to 11.26�C,while with increase in ethanol concentration from 0% to 30% vol/vol, the TP value decreases from 14.13�C to 8.66�C.6 These resultsindicate that a fine balance is required between cholesterol andethanol in order to achieve ethosome vesicles with higher elasticitywhile maintaining the physical stability.

Researchers have also studied the influence of lipid and ethanolcontent on physicochemical properties of ethosomes such as

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-2310

vesicle size and morphology, entrapment efficiency, zeta potential,and elasticity. Transmission electron microscopy study on etho-somes indicated that they are relatively imperfect round shapevesicles owing to fluidizing effect of ethanol on lipid bilayercompared to ultradeformable liposome.124 In our investigation ondiclofenac-loaded ethosomes, we found that with increase inethanol concentration from 0% to 30%, the vesicle size decreased,elasticity increased, and zeta potential decreased. On the otherhand, entrapment efficiency increased with increase in the ethanolconcentration from 0% to 20% due to its cosolvent effect.6,125

However, entrapment efficiency decreased with further increasein ethanol concentration, probably due to excessive vesicularfluidization causing leakage of the drug. Similar results wereobserved by other researchers.126 Additionally, among studiedproperties (i.e., vesicle size, elasticity, zeta potential, and entrap-ment efficiency), vesicle size and elasticity of the ethosomes wereidentified as the only 2 dominating physicochemical propertiesthat affect the skin permeation of the ethosomes. These physico-chemical properties could be suitably manipulated by modificationof formulation variables (PC:CH ratio and ethanol) to achievedesired therapeutic permeation flux.6

Presence of ethanol also provides a net negative surface chargethat prevents aggregation of the vesicles due to electrostaticrepulsion. In a study, colloidal stability of liposomes and ethosomeswere evaluated using Turbiscan optical analyzer. It was found thatcompared to liposomes (Phospholipon 100G®, cholesterol, andwater), ethosomes (Phospholipon 100G, ethanol, and water)showed no coalescence, sedimentation, and flocculation indicatingsuperior physical stability.127 In another study, the econazolenitrate-loaded ethosomes (soy phosphatidylcholine, ethanol, andwater) was found to be physically stable for 6 months under thetested condition 25�C.128

Although the presence of ethanol in the ethosomes can enhanceskin permeation, it can also lead to skin irritation. To address thisconcern, few researchers have studied the impact of ethosomalvesicles on skin morphology. Buspirone-loaded ethosomes with38% ethanol exhibited no change in skinmorphology. The thicknessand appearance of the horny layer were found to be unchanged incomparison to the normal untreated rat skin.129 Similar results onhuman skin were also reported in other studies.130,131 In general,ethanol concentration of approximately 30%-40% is consideredwidely acceptable for skin delivery via ethosomes.

Several studies have been conducted to explore the applicationof ethosomal delivery for variety of drugs.3,128,132-135 Table 2 sum-marizes the recent patents on the application of ethosomal drugdelivery. There are several reported studies of superior skin de-livery of ethosomes compared to liposomes, ultradeformable lipo-somes, andmarketed formulations. Psoralen-loaded ethosomes (anantipsoriasis drug) has shown 3.50 and 2.15 times higher perme-ation flux and skin deposition respectively, compared to that ofliposomes.136 Ethosome-mediated apigenin delivery produced amore prominent effect on UVB-induced skin inflammation bysuppressing COX-2 levels, compared to liposomes and deformableliposomes.137 Ethosomal formulation of 5-aminolevulinic acid alsoshowed better skin permeation than liposomes in photodynamictherapy.138 Anticandidal activity against Candida albicanswas foundto be highest for clotrimazole-loaded ethosomal formulation withthe highest zone of inhibition (34.6 ± 0.57 mm), in contrast todeformable liposomal formulation (29.6 ± 0.57 mm) and marketedcream formulation (19.0 ± 1.00 mm).124 The superior anticandidalactivity of the ethosomes was attributed not only to the obviouspermeation enhancement effect of these vesicles but also to thepresence of ethanol that has potential to kill organisms by dena-turing their proteins. Similarly, healing time in herpes infectedpatients was significantly improved by acyclovir-loaded ethosomal

cream in comparison to the market formulation.139 In anotherexperiment, ammonium glycyrrhizinate-loaded ethosomes elicitedan increase in in vitro percutaneous permeation (in human skin)and anti-inflammatory activity (in human volunteers) compared tothe ethanolic or aqueous solutions of this drug.131 For diclofenac-loaded ethosomes, the permeation flux of the optimized formula-tion was 12.9 ± 1.0 mg/h$cm2, which was significantly higher thanthe drug-loaded conventional liposome, ethanolic or aqueous so-lution.6 Furthermore, in vivo pharmacodynamic study indicatedthat optimized ethosomal hydrogel exhibited enhanced anti-inflammatory activity (reduction in paw edema volume)compared with liposomal and plain drug hydrogel formulations. Inorder to improve the skin permeation of ethosomes, researchershave explored various interesting arena. For example, low fre-quency ultrasound was utilized to deliver hydrophilic macromol-ecules, hyaluronic acid (MW 1500 kDa). In vitro permeation studyrevealed that the combination of low frequency ultrasound andethosomes improves the permeation enhancing effect for hyal-uronic acid by 2.1 times and 6.4 times compared to ethosomesalone and hyaluronic acid solution with low frequency ultrasound,respectively.140

Furthermore, some promising results are also observed in thehormonal therapy. In a study, the skin permeation potential of thetestosterone-loaded ethosomes was compared with the marketedtransdermal patch of testosterone. The authors observed nearly 30times higher skin permeation of testosterone from the ethosomalformulation compared to the marketed formulation.130 Touitouet al. tested the effect of an ethosomal insulin formulation on theblood glucose level that was applied to the skin. The ethosomalformulation instigated up to 60% decrease in blood glucose levels inboth normal and diabetic rats and the level was maintained for atleast 8 h.25

Some researchers have also compared the effectiveness ofethosomal delivery via transdermal route with that of the drugdelivery via oral route. In vivo pharmacokinetic study of ethosomaltransdermal therapeutic system showed approximately 3 timeshigher bioavailability compared with oral suspension of valsar-tan.141 Transdermal application of buspirone-loaded ethosomes formenopausal syndromes showed similar Cmax value compared to theoral aqueous solution (120.07 ± 86.97 and 93.44 ± 76.46 ng/mL,respectively).129 Furthermore, buspirone delivered via transdermalroute was present for longer time period compared to the oraladministration (12 vs. 4 h, respectively), suggesting more sustainedand nonfluctuated delivery to plasma with reduced side effects viatransdermal application of buspirone-loaded ethosomes.129

Moreover, there have been various reported studies exploringthe mechanism of ethosomal delivery via skin. Study on theamphiphilic fluorescent probe D-289 containing trihexyphenidylhydrochloride ethosomes has suggested that ethosomes permeatedeeper into the skin compared to classic liposomes (about 170 and96 mm respectively from the skin surface).142 Recently, CLSM studyalso indicated that “intact” ethosomes were able to reach deeperstratum corneum layers due to higher elasticity of these vesicles.143

Skin permeation behavior via ethosomes is generally attributed tothe solvent properties of ethanol followed by “ethosome”effect.26,58 Figure 4 shows the diagrammatic representation of thismechanism. The stratum corneum lipids, at physiological temper-ature, are densely packed and highly ordered. Ethanol in ethosomesinteracts with the polar head group region of the lipid molecule,resulting in a reduction in the transition temperature of the stratumcorneum lipids and consequently increasing their fluidity. Ethoso-mal vesicles containing drug, owing to the smaller vesicle size andhigh elasticity, can then permeated through the partially fluidizedskin lipids to deliver the drug into deeper skin layers. In a study,CLSM has demonstrated enhanced skin permeation of ethosomes

Table 2Some of the Recent Patents on Ethosome Delivery System

Application Title Inventors Filling Year Results

CN 104706571 A Preparation method of ethosome/natural material/polyvinyl alcoholcomposite hydrogel

Yang Xingxing, Lynn, Chen Mengxia,Fanlin Peng

2015 Addition of the polyvinyl alcohol improvedthe mechanical properties of thehydrogel

CN103536700 A Chinese medicinal ethosome gel patchfor treating herpes zoster andpreparation method thereof

Bu Ping, Hu Rong, Chen Lin, Wei Rong,Wu Huanhuan, Huang Xiaoli

2014 Easy in medication and convenient to use,has a good therapeutic effect, quickresponse, strong analgesic action but noadverse reaction

CN103893394 (A) Ethosome gel film-coating agent withmultiple wound repair effects andpreparation method of ethosome gelfilm-coating agent

Chen Jie, Huang Changping, Zheng Maoxin,Nie Kaipin

2014 The ethosome entrapped film-coating agenthelps to promote healing and nutritionsupplying of the wound tissue. Theethosome gel film-coating agent issuitable for wound clinical care andtreatment

CN103800277 (A) Leflunomide ethosome compositionand its preparation method

Zhang Tao, Ding Yanji, Deng Jie, Luo Jing,Zhong Xiaodong

2014 Improves the transdermal rate ofleflunomide, can significantly reduce sideeffects of oral administration ofleflunomide, and improves curativeeffects

EP 2810642 A1 Chitosan-modified ethosome structure Chin-Tung Lee, Po-Liang Chen 2013 The chitosan-modified ethosome structurecontains active substances with differenteffects, such that it improves the storageand transportation of multiactivesubstances

CN103006562 (A) Daptomycin ethosome preparation Li Chong, Liu Xia, Yin Qikun,Wang Xiaoying,Chen Zhangbao

2013 The daptomycin ethosome preparation is astable translucent dispersion systemwith light blue opalescence, small anduniform in particle size, high inentrapment efficiency and excellent intransdermal performance, drug releaseand has certain slow-release effect, andthe preparation method is simple andconvenient, low in cost and good instability

CN102688194 B Preparation method of lidocaineethosome

Liang Ju, Wu Wenlan, Li Mei, Miao Juan,Wei Xuefeng, Chen Shan, Wang Xiao taro

2012 The method obtained lidocaine ethosomesstable, high encapsulation efficiency,process optimization encapsulationefficiency up to 80.93%. Lidocaineethosomes good compatibility with theskin

CN102552147 (A) Bullatacin ethosome gel andpreparation method thereof

Jianping Tan, Lixin Jiang, Tanran Chang,Zhiwen Zhou

2012 The bullatacin ethosome gel provided bythe invention can reduce irritation to theskin and has good percutaneouspenetration effects

CN102813624 (A) Lidocaine ethosome and preparationmethod thereof

Zhao Xianying, Su Yongping, Gao Jining,Liu Yimin, Zhao Huawen, Xiao Xiang,Zhou Xiaoxia, Zhang Dinglin, Wu Liping

2012 The lidocaine ethosome of the presentinvention provides advantages of rapidonset, prolonged drug action time,further has advantages of small particlesize, high penetration efficiency, highencapsulation efficiency

CN102579323 (A) Paclitaxel ethosome gel andpreparation method thereof

Jianping Tan, Lixin Jiang, Tanran Chang,Zhiwen Zhou

2012 The action of stimulation to the skin can bereduced, and the percutaneouspermeation effect is good

CN102397255 (A) Progesterone ethosome, andpreparation method and applicationthereof

Shu Zhang, Hong Deng, Huaqing Lin,Xiaoling Zhang

2012 The progesterone ethosome is mainlyapplied to hormone replacementtherapy, secondary amenorrhea,functional aplastic bleeding, andpremenstrual syndrome

CN102133183 (A) Acyclovir ethosome and preparationmethod thereof

Xuewen Wu, Yan Xiong 2011 Acyclovir ethosome has high stability andnarrow particle size distribution

CN102144972 (A) Podophyllotoxin ethosomes andpreparation methods thereof

Nianping Feng, Yanyan Yu, Jihui Zhao,Haiting Weng, Xiaoqin Shi

2011 The aims of increasing curative effect andreducing relapse and toxic side effectsare fulfilled. The invention also discloses2 preparation methods for thepodophyllotoxin ethosomes

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-23 11

compared to liposomes, with permeation up to the last layer ofepidermis (to stratum basale).128,141 In another interesting study,CLSM revealed the presence of “intact de-shaped” vesicles pene-trating through the skin, suggesting that these vesicles squeezethemselves to deeper skin layers owing to their high elasticnature.143

A study using double staining technique indicated that etho-somes entered the skin between the coreocytes through theintercellular lipid domain.144 FTIR studies indicated that mildswelling of corneocytes and skin lipid fluidization (penetrationpathways) were observed with ethosomal formulation.124 Howev-er, ethosomes were found to deliver drug to deeper skin layers

Figure 4. Mechanism of skin permeation via ethosomes.

Table 3List of Emerging Lipid Vesicles for Skin Drug Delivery

Emerging LipidVesicles

Definition Reference

Archeosomes Archeosomes are vesicles consisting ofarchebacteria lipids, which are chemicallydistinct from eukaryotic and prokaryoticspecies. They are less sensitive to oxidativestress, high temperature, and alkaline pH

147,148

Lipoplexes Cationic lipid-DNA complexes, named lipoplexes,are efficient carriers for cell transfection buthave certain drawbacks due to their toxicity.These toxic effects may result from eithercationic lipids or nucleic acids

149

Proliposomes Proliposomes are defined as dry, free-flowingparticles that immediately form a liposomaldispersion on contact with water

150-152

Cubosomes Cubosomes are discrete, submicron,nanostructured particles of bicontinuous cubicliquid crystalline phase

153-155

Ufasomes Ufasomes containing lipid carriers that attachedto the skin surface and allows lipid exchangebetween the outermost layers of the stratumcorneum

156,157

Niosomes Niosomes are nonionic surfactant andcholesterol-based vesicle with improvedstability than liposomes (especially oxidativestability due to absence of phosphatidylcholinein niosomes)

158,159

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(dermis) via hair follicular route in another reported study.145 It isstill unclear whether ethosomes transport mechanism involvesintracellular route or follicular route or both.

Ethosomes have provided a new frontier in the field of vesicularskin delivery. However, based on the literature review, we feel thatsome key issues are not addressed in this field (Table 1). Forexample, although short-term skin toxicity of ethanol (in etho-somes) is available in the literature, but long-term effects ofrepeated applications (clinically relevant dosing) of ethosomalformulation is not studied. Also, long-term structural and chemicalstability during storage is not investigated in the systematicmanner. Finally, scalability of the manufacturing process for etho-some is not available in the literature.

Emerging Lipid VesiclesThe utility of various lipid vesicles has encouraged researchers

to modify these vesicle to impart specific structural or applicationproperties.146 Table 3 lists these new emerging lipid vesiclesdeveloped in recent past for drug delivery. Current research onthese vesicles is rather limited, especially for topical drug deliveryand not in the scope of this review article. Interested readers aredirected to the references mentioned in Table 3.

Lipid Particulate Systems

In the recent years, the lipid particulate systems have gainedhuge popularity as an alternative to lipid vesicular delivery systemssuch as liposomes, ultradeformable liposome, and ethosomes.160,161

Some of the currently marketed products based on lipid-based de-livery systems are listed in Table 4. Additionally, compared to poly-meric nanoparticles, lipid particulate systems are preferred due toavailability of biocompatible and nontoxic lipid excipients forfabrication of these delivery systems.162 Lipid particulate systems

typically include lipospheres and lipid nanoparticles such as SLNsand NLCs. SLNs can be considered as the first generation of lipidnanoparticles, whereas NLCs are regarded as the second-generationlipid nanoparticles overcoming the shortcomings of SLNs. Table 1summarizes the information on lipid particulate delivery systems.

LipospheresLipospheres are water dispersible solid microparticles with

particle size range of 0.2-500 mm.163 It consists of a solid hydro-phobic lipid core stabilized by a monolayer of phospholipidembedded on the surface. Some of the benefits of liposphere drugdelivery are improved drug stability, possibility for extendedrelease of entrapped drug, controlled particle size, high drugloading mainly for hydrophobic drugs, high dispersability in anaqueous medium, low cost of ingredients, ease of preparation, andscale-up.27 Lipospheres have been reported to enhance the pene-tration of drugs through the stratum corneum for variety of drugsby forming an occlusive film on the skin surface.28

Several techniques such as melt dispersion technique, solventemulsification evaporation, solvent emulsification-diffusion tech-nique, hot and cold homogenization, multiple microemulsionmethod, ultrasonication or high-speed homogenization, and high-pressure homogenization have been used for the production oflipospheres.27 For hydrophobic core of the lipospheres, naturallyoccurring lipids such as triglycerides, waxes, or fatty acids are used.In addition, neutral fats and stabilizers are also used in the prepa-ration of the hydrophobic core. Some of the phospholipids that areused to form the surrounding layer of lipospheres include soybeanphosphatidylcholine, pure egg phosphatidylcholine, phosphati-dylethanolamine, dimyristoyl phosphatidylglycerol, and food gradelecithin.27 Depending on the physicochemical properties, the drugis either dissolved or dispersed in a solid fat matrix. Hydrophilicdrugs have shown lower entrapment in such lipids. However, polarlipids such as cetyl alcohol, stearyl alcohol, and cetostearyl alcoholhave been utilized to successfully overcome this limitation.27,164

Entrapment efficiency and particle size are considered pre-dominant physicochemical properties that influence the skin de-livery potential of the lipospheres. Type of lipid, amount of

Table 4Currently Marketed Cosmetic Products with Lipid-Based Delivery Systems

Trade Name Manufacturer

LiposomeRovisome ACE Plus ROVI Cosmetics

International GmbHAgeless Facelift cream I-Wen NaturalsAmeliox Mibelle BiochemistryAstraForceLipobelle GlacierNano-Lipobelle S100/PAPhytoCellTec™Revitalift L’OrealLancome Soleil Soft-Touch Anti-WrinkleSun Cream SPF 15

L’Oreal

EthosomesCelltight EF Hampden HealthDecorin cream Genome CosmeticsNanominox SinereNoicellex Novel Therapeutic

TechnologiesSkinGenuity Physionics

Lipid nanoparticlesCutanova Cream Nano Repair Q10 Dr. RimplerIntensive Serum Nanorepair Q10Cutanova Cream Nanovital Q10SURMER Cr�eme Leg�ere Nano-Protection Isabelle LancraySURMER Cr�eme Riche Nano-RestructuranteSURMER Elixir du Beaut�e Nano-VitalisantSURMER Masque Cr�eme Nano-HydratantNanoLipid Restore CLR Chemisches LaboratoriumNanolipid Q10 CLR Dr. Kurt Richter (CLR)Nanolipid Basic CLRNanolipid Repair CLRIOPE SuperVital line of- AmorePacificCreamSerumEye creamExtra moist softenerExtra moist emulsion

NLC Deep Effect Eye Serum Beate JohnenNLC Deep Effect Repair CreamNLC Deep Effect Reconstruction CreamRegenerationscreme Intensiv SchollSwiss Cellular White IlluminatingEye EssenceSwiss Cellular White Intensive Ampoules

La Prairie

SURMER Creme Contour Des Yeux Nano-Remodelante

Isabelle Lancray

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-23 13

phospholipid, method of preparation, and concentration of stabi-lizer are some of the factors that affect the entrapment efficiency oflipospheres.29 In general, smaller lipospheres are preferred toimprove skin penetration over larger lipospheres. Small size of thelipospheres (especially in submicron range) ensures close contactto stratum corneum and can also increase the amount of drugpenetrating into the mucosa or skin layers.29

With an emergence of lipid nanoparticles (SLNs and NLCs), theresearch involving application of lipospheres for skin drug deliveryhas been limited. Major research utilizing lipospheres for skin drugdelivery focuses on the anti-inflammatory drugs such as benzo-caine, flurbiprofen (FP), and aceclofenac165-167; photolabile drugssuch as melatonin and UV filters168,169; and protein and peptide.170

In one of the reported studies, aceclofenc lipospheres preparedusing tristearin to phosphatidylcholine weight ratio of 2:1 exhibi-ted superior anti-inflammatory activity compared to the marketedproduct in rat paw edema test.167 The result was attributed to theocclusive film forming ability of the lipospheres. The solid matrix ofthe lipospheres can also protect photo or thermal labile drugsagainst physical and chemical degradation. In one study, the effectof formulation components on the physicochemical properties and

shielding efficiency of the photo labile drug-loaded lipospheres wasinvestigated.168 Lipospheres loaded with melatonin were preparedusing tristearin or tripalmitin as the lipid core and hydrogenatedphosphatidylcholine or polysorbate 60 as the emulsifier. It wasobserved that the liposphere yield was significantly affected by thelipid/emulsifier ratio with the highest yield obtained for triglycer-ide/emulsifier ratio of 3:1.168 In addition, the photolysis experi-ments demonstrated that the light-induced decomposition ofmelatonin was markedly decreased by encapsulation into lipidmicrospheres based on tristearin and phosphatidylcholine (theextent of degradation was 19.6% for unencapsulated melatonincompared to 5.6% for the melatonin-loaded microparticles). Theseresults indicate that lipospheres can provide an effective strategy toenhance the photostability of melatonin.168 In another study, in-clusion complex between HP-b-CD and butyl methoxydibenzoyl-methane (BMDBM, the sunscreen agent) was loaded into thelipospheres to study the influence of this system on sunscreenphotostability. BMDBM/HP-b-CD complex was prepared andloaded in melted lipids during liposphere preparation.171 Release ofBMDBM from the lipospheres was lower when it was incorporatedas inclusion complex rather than as a free molecule. The photo-degradation studies showed that complex-loaded liposphere sys-tem achieved a significant reduction in light-induceddecomposition of the free sunscreen agent (the BMDBM lossdecreased from 28.9% to 17.3%-15.2%).171

Although aforementioned studies indicate successful application oflipospheres, the insufficiently reported physical stability data are aconcern. Furthermore, relatively higher particle size of lipospheresposes challenges during skin delivery. Also, emergence and advance-ment of lipid nanoparticles has drawn researchers away from lipo-spheres due to obvious advantage of nanocarriers overs microcarriers.

Solid Lipid NanoparticlesSLNs are colloidal drug delivery systems composed of physio-

logical and biodegradable lipids.172-174 These lipids form a solidlipophilic matrix at the room temperature in which hydrophilic orlipophilic drugmolecules can be incorporated (Fig. 5). Typically, thelipid content ranging from 1% to 30% wt/wt and surfactant con-centration ranging from 0.5% to 5% wt/wt is used. Structurally, theyare spherical in shape with an approximate mean particle size inthe range of 50-1000 nm and usually yield narrow particle sizedistribution around the mean particle size.

SLNs are widely studied for therapeutic efficacy via skin deliveryroute. Compared to lipid-based vesicular carriers, SLNs provideflexibility in modulating the drug release, higher drug loading oflipophilic moieties, and enhance drug stability by protecting thedrugs from chemical degradation, oxidation, light degradation, andmoisture (Table 1). Due to small particle size and consequentlyhigher surface area, these nanoparticles achieve close contact withsuperficial junction of corneocyte clusters and channels of stratumcorneum.172 This is particularly important to improve drug accu-mulation and local drug depot formation, which can be utilized forcontrolled delivery of the drug over a period of time. SLNs alsopossess a distinct occlusive property, which may enhance thepenetration of drugs through stratum corneum by decreasingtransepidermal water loss. Due to higher water content of SLNs,lipid nanoparticle dispersions are now incorporated intocommonly used dermal carriers (e.g., gels or creams) such as car-bopol gel and hydrogel to obtain semisolid formulations.175-177

Furthermore, it has been reported that SLNs enhance the pene-tration and transport of active substances, particularly lipophilicagents, and therefore intensify the concentration of these agents inthe skin.30,161,172

In addition, the manufacturing processes of SLNs are costeffective, reproducible, and scalable. Manufacturing processes

Figure 5. Structural difference between SLN and NLC.

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-2314

utilized to prepare SLNs include high shear homogenization, high-pressure homogenization (hot and cold homogenization), solventemulsification and evaporation or diffusion, microemulsion, w/o/wdouble emulsion method, and high-speed stirring and sonicat-ion.30,178 High-pressure homogenization has been accepted as areliable and effective technique for the preparation of SLNs withbetter submicron nanoparticles than high shear mixing or ultra-sound.179 Furthermore, high-pressure homogenization techniquehas also shown a good scalability and feasibility for SLNmanufacturing.30 Most of the preparation techniques have reportedto result in solid lipid matrix type structure from which drugrelease occurs by diffusion.160,172,180,181

Several reports have indicated that formulation composition canbe suitably tailored to modify the physicochemical properties thatcan lead to effective drug delivery via skin.162,172 Based on literaturereview, particle size and entrapment efficiency of SLNs are mostrelevant parameters controlling the effectiveness of the drug de-livery. In one of the reported studies, SLNs with different particlesize (80, 333, and 971 nm) were prepared using hot melt homog-enization technique using Precirol as solid lipid and Rhodamine Bas fluorescent dye. Effect of particle size on skin permeation wasthen studied in rat skin using fluorescent microscopy. The resultsindicated size-dependent skin permeationwith lowest particle sizeSLNs exhibiting the highest skin permeation. Based on these find-ings, authors suggested that sub 100 nm size range is optimal forskin delivery of SLNs, possibly via hair follicular route.182 In thisregard, the type of lipid used for preparation of SLNs plays a crucialrole.30,172 For example, using the hot homogenization, it has beendemonstrated that lipids with higher melting point result in higherparticle size of SLNs.162,183 SLNs prepared with same concentration(5%) of either steric acid (low melting lipid) or Compritol ATO 888(high melting lipid) resulted in particle size of 50 and 80 nm,respectively.183 Other than lipids, selection of surfactants and theirconcentrations has also shown significant impact on the physico-chemical properties of SLNs. It was found that the use of lipid-basedsurfactants (Labrasol or Labrafil) enhances the solubility of thelipophilic drug and thus increases its entrapment efficiency in theSLNs.160 In another study, the effect of Poloxamer F-68, PoloxamerF-127, and Tween 80 on the physicochemical properties ofAmphotericin B-loaded SLNs was evaluated. It was observed thatfor drug:lipid ratio of 1:10, compared to Poloxamer F-68 and Tween80-based SLNs, Poloxamer F-127-based SLNs exhibited lowestparticle size (242.0, 373.0 vs. 111.1 nm, respectively), higher zetapotential (�12.64, �6.12 vs. �23.98, respectively) and higher

entrapment efficiency (86.4%, 81.9% vs. 93.8%, respectively).184 In aninteresting study, the effect of 3 independent factors, that is, theconcentration of lipid, surfactant, and drug on the response vari-ables (particle size and entrapment efficiency) of fluocinoloneacetonide (FA)-loaded SLNs, was studied using 3-factor, 3-levelBox-Behnken design. It was concluded that the concentration oflipid had positive effect while concentration of surfactant hadnegative effect on the particle size of SLNs (particle size in the rangeof 99.26-132.66 nm). On the other hand, all 3 independent factors,that is, concentration of lipid, surfactant, and drug, had positiveeffect on entrapment efficiency of SLNs (entrapment efficiency inthe range of 67.28%-88.79%).185

Major reported research on SLNs include topical delivery of (1)antifungal agents such as amphotericin B,184 griseofulvin,180 andterbinafine hydrochloride175; (2) antioxidants such as hydroqui-none,186 idebenone (IDB),187 and isotretinoin188,189; (3) drugs forskin diseases such as adapalene,190 psoralen,31 and curcumi-noids191; (4) drugs for treatment of chronic wounds192,193; (5)nonsteroidal anti-inflammatory drugs (NSAIDS) such as melox-icam,194 dexflurbiprofen,195 and ketoprofen196; and (6) glucocorti-coids such as betamethasone dipropionate197 and FA.185

In in vitro cytotoxicity studies on human keratinocyte cells(HaCaT), griseofulvin-loaded lipid nanoparticles (GF-LN) have re-ported a better safety profile compared to pure griseofulvin sus-pension.180 In addition, GF-LN demonstrated comparableantifungal activity against Trichophyton rubrum (minimum inhibi-tory concentration 0.5 mg/mL) and Trichophyton mentagrophytes(minimum inhibitory concentration 0.25 mg/mL), indicating no lossof antifungal activity after being incorporated in LN. Furthermore,higher drug accumulation was reported in upper layers of the skin,which may minimize diffusion of drug from the dermis layer intothe systemic circulation. Amphotericin B-loaded SLN dispersion gelalso demonstrated similar drug localizing effect in the skin withimproved antifungal activity.184 Similarly for glucocortioids, in vitroskin distribution studies showed the presence of significantamount of FA on the epidermal layer of skin when treated with FA-loaded SLN suspension compared to plain FA suspension. Further-more, drug release study confirmed prolonged release from theSLNs following Higuchi release kinetics with R2 value of 0.995.185

As mentioned above, another interesting area of research is thetreatment of wound healing. In a study, Astragaloside IV-loadedSLNs were prepared using the solvent evaporation method andfurther incorporated in carbomer hydrogel to study wound healingand antiscar effects by topical route. The wound scratch test and

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-23 15

lipid nanoparticle uptake study via skin cells revealed enhancedmigration and proliferation of keratinocytes with increased druguptake on fibroblasts through the caveolae endocytosis pathway.Moreover, Astragaloside IV-based SLN gel strengthened woundhealing and inhibited scar formation in vivo by increasing woundclosure rate and by contributing to angiogenesis and collagenregular organization.198

SLNs are also reported to be an excellent carrier for loading ofantioxidants as well. It is well known that the photochemicalinstability of these compounds has been a limiting factor for theirskin applications. In a study, stearyl ferulate-based solid lipidnanoparticles (SF-SLNs), as vehicles for b-carotene and a-tocoph-erol, were formulated to improve the stability of these compounds.Ferulic acid (by-product of stearyl ferulate) is a potent antioxidanthaving synergistic effects with other antioxidants (e.g., b-caroteneand a-tocopherol) and it is able to protect and stabilize them fromdegradation. SF-SLNs were demonstrated to provide a good vehiclefor b-carotene and a-tocopherol by preventing oxidation anddegradation of both compounds.199

Despite the aforementioned success in skin drug delivery, lowdrug-loading capacity (especially hydrophilic drugs) and drugexpulsion during storage have caused major challenges for thisdelivery system. Additionally, SLNs can undergo a rapid, unpre-dictable, and irreversible gelation phenomenon, where low-viscosity SLN dispersion can transform into viscous gel during thecooling of dispersion.32 Gelation can also result in increase in par-ticle size and particle agglomeration.32 Furthermore, SLNs aremanufactured mostly in dispersion form and therefore are requiredto be incorporated in semisolid carriers like gels or ointments.Physical stability of SLNs can also cause issues during shelf life.However, recent studies have suggested that SLNs can be stored at4�C without affecting its physicochemical properties.184,185 Simi-larly, FA-loaded SLNs showed no significant changes in particle size,zeta potential, and entrapment efficiency when stored at 4�C for 3months.185 Also, amphotericin B-loaded SLNs exhibited a goodstability over the period of 3 months storage (at 2�C-8�C and 25�C)with no significant change in clarity and any phase separationthrough visual examination.184

Nanostructured Lipid CarriersAs discussed above, SLNs’ success in topical drug delivery is

mainly limited by poor drug loading, risk of gelation, particleagglomeration, and drug leakage during storage. In order to over-come the potential challenges of SLNs, the second generation oflipid nanoparticles, namely NLCs, is now widely studied.31,33,200

NLCs have been introduced for pharmaceutical and cosmeticapplication with more than 40 products currently available in thecosmetic market.200 Its commercial success is mainly attributed tohigh drug loading, biodegradable components, prevention or

Table 5Comparison of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers

Parameters Solid Lipid Nanoparticles

Composition SLNs are composed of 1% (wt/wt) to 30% (wt/wt) solid lipdispersed in an aqueous medium and if necessary stabwith preferably 0.5% (wt/wt) to 5% (wt/wt) surfactant

Microstructure Highly ordered lipid matrixPreferred method of

preparationHigh-pressure homogenization

Drug loading Due to its high degree of order, the number of imperfectithe crystal lattice is reduced leading to drug expulsion

Occlusivity HigherMechanism of enhancing

skin permeationDue to small particle size and consequently higher surfac

these nanoparticles achieve close contact with superfijunction of corneocyte clusters and channels of stratumcorneum

minimization of active ingredients expulsion, avoidance of organicsolvents during the preparation, and suitability for large-scaleproduction by using existing technologies.

Preparation techniques and components for NLCs are similar toSLNs.However, unlike SLNs,NLCs are producedbymixing at least oneliquid lipid (oils) with the solid lipid(s) to formnanocapsule inwhichthe liquid lipid phase can be embedded into the solid matrix or to belocalized at the surface of solid particles.34,201 In literature, the typicalweight ratio of solid lipid to liquid lipid ranges from 70:30 to 90:10.Mixing of liquid and solid lipids induces a melting point depressioncompared to thepure solid lipid. The resultingstructure remains solidat room temperature with API-loaded liquid pocket.34,202,203 Incor-porating oil in the solid lipid matrix distorts the lipid crystals bycreating imperfections in the lattice, which facilitate higher drugloading (Fig. 3). NLCs also minimize drug expulsion during storageand possess less water content unlike SLN dispersions.204 Table 5provides the comparison between SLNs and NLCs.

The impact of formulation variables on the particle size andentrapment efficiency of the NLCs has also been studied. Forminoxidil-loaded NLCs, it was demonstrated that the ratio of solidlipid (tristearin) to liquid lipid (oleic acid) could be suitably modi-fied to achieve smaller particle size, higher entrapment efficiency,and improved physical stability.205 In another study, the effect ofconcentration of lipid, concentration of surfactant, and concentra-tion of drug was studied on the particle size and entrapment effi-ciency using 3-factor, 3-level Box-Behnken design. It was observedthat the ratio of liquid lipid to total lipid and concentration of drughas positive effect while concentration of surfactant has negativeeffect on the particle size of NLCs. On the other hand, the ratio ofliquid lipid to total lipid, concentration of surfactant, and concen-tration of drug has positive effect on the entrapment efficiency ofNLCs. Furthermore, release study indicated prolonged drug releasefrom the NLCs following Higuchi release kinetics and zero-orderrelease kinetics.206

Mechanism of action of NLCs in the topical drug delivery issimilar to that of the SLNs. Smaller size of NLCs improves surfacecontact to the stratum corneum and consequently increases theamount of active compound penetrated through the skin. In addi-tion, nano-sized particles can tightly adhere to the skin surface andallow the delivery of drug in a more controlled fashion. Further-more, because NLCs provide higher drug loading than SLNs, it canachieve high drug concentration gradient on the skin surface tofacilitate drug permeation.4,207

NLCs have been successfully utilized to deliver the drugs viatopical route for improving drug permeation, skin hydration,controlled drug release, and drug stability.150,208,209 In recent past,topical delivery of clotrimazole,210 psoralen,31 enoxaparin,211

lutein,212 and CoQ10-loaded NLCs213 has been reported. In astudy, the effect of NLCs, nanoemulsion (NE), or oil solution on the

Nanostructured Lipid Carriers

idilized

Blends of solid lipids and liquid lipids (oils), preferably in a weight ratioof 70:30 up to a ratio of 90:10

Less ordered lipid matrixHigh-pressure homogenization

ons in By blending solid and liquid lipids, a less ordered lipid matrix is createdwith higher drug load potential

Lowere area,cial

Similar to SLNs. Due to small particle size and consequently highersurface area, these nanoparticles achieve close contact withsuperficial junction of corneocyte clusters and channels of stratumcorneum

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-2316

permeation of IDB (a synthetic antioxidant) was evaluated usingex vivo guinea pig skins. It was observed that for NLC formulation,the cumulative amount of IDB in the epidermis, dermis, andacceptor medium of diffusion cells was approximately 3-foldhigher than the NE or oil solution at the end of 24 h experiment.Furthermore, the stability of NLCs and NE was also evaluated bymeasuring their diameter, zeta potential, and entrapment effi-ciency after 30, 90, and 180 days of storage at 25�C in dark, 40�C indark, and 25�C under daylight, respectively. NLCs exhibited supe-rior physical stability at all storage conditions compared to NE.214

Thus, NLCs not only showed better permeation profile but alsoimproved physical stability during storage. Researchers have alsoreported NLC-loaded gel as a ready to use topical delivery systemswith no adverse effect on the properties and behavior of NLCs.4,215

In one of the reported study for delivery of NSAIDs, FP (for treat-ment of arthritis)-loaded NLCs were prepared by the optimized o/wemulsification homogenization-sonication technique. Differentialscanning calorimetry of the treated skin indicated that the NLCspenetrate into follicles of the skin and accumulate in the dermisand consequently improved bioavailability.216 The in vivo evalua-tion revealed 1.7-fold improved bioavailability to that of commer-cial gel. Similarly, FP-loaded NLCs were prepared by hot high-pressure homogenization method with Compritol® ATO 888,Miglyol® 812, lecithin, FP, and aqueous surfactant solutions ofPoloxamer 188 and sodium deoxycholate. NLCswere then loaded inCarbopol (FP-NLC-gel) and compared with FP-loaded gel throughrat skin. NLC gel exhibited pseudoplastic flow with thixotropybehavior, which is essential for topical drug delivery.217 Further-more, FP-NLC-gel showed a more pronounced permeation profilecompared to FP-loaded common gel through rat skin. Themaximum concentration in plasma was 29.44 and 2.49 g/ml afteroral (FP methylcellulose suspension) and transdermal (FP-NLC-gel)administration, respectively. Lower plasma level exposure of FP viatransdermal delivery suggests lower systemic (especially gastro-intestinal) side effects, which is commonly associated with oraldelivery of NSAIDs like FP.5

In addition to improved dermal delivery, NLCs were explored toprovide protection for photolabile drugs. In one of such studies,photolabile alpha-lipoic acid (ALA-antioxidant)-loaded NLCs wereprepared with hot high-pressure homogenization technique. FreeALA and ALA-loaded NLC aqueous dispersion (composed of glycerinmonostearate, glyceryl triacetate, and glyceride) was exposed tonatural daylight and improvement in ALA photostability was vali-dated by evaluating the percentage of retained ALA under the naturallightexposure. FreeALA (dissipated inmethanol) degradedmore than99% of its original concentration under natural daylight in 4 months,while ALA-NLCs allowed ALA retention up to about 88.5% under thesame conditions. This finding suggests potential use of NLCs as aneffective alternative to improve the photostability of various com-pounds utilized in nutrition, dermal and cosmetic applications.218

Modified NLCs have also been studied to further exploit andexpand the utility of NLCs for topical drug delivery, especially forskin disorders including psoriasis, atopic dermatitis, and allergiccontact dermatitis.219,220 In an interesting study, NLCs were pre-pared by modified hot melt homogenization technique to load2-model anti-inflammatory drugsdspantide II and ketoprofen,respectively. NLCs were further taggedwith polyarginine peptide toimprove skin permeation of the actives. Surface modified NLCsshowed enhanced skin permeation of spantide II and ketoprofen tothe deeper skin layers (viable epidermis and dermis) and conse-quently reduced ear swelling associated with allergic contactdermatitis. Authors also claimed that these results could be appli-cable to various other skin disorders like psoriasis, fungal, bacterial,viral infections, and skin cancers like melanoma.220 In anotherstudy, Tacrolimus (poorly soluble drug)-loadedmodified NLCs were

prepared using lipophilic solubilizer in place of liquid lipids.221

Tacrolimus was dissolved in a minimum amount of lipophilicsolubilizer (propylene glycol monocaprylate) before preparingNLCs with high-pressure homogenization technique with glyceryltrimyristate as solid lipid. Delivery of Tacrolimus-NLC-enriched gelsshowed significantly higher in vitro drug release, skin permeation,and in vivo bioavailability compared to commercial ointment.Furthermore, in vivo gamma scintigraphy also revealed thatradioactivity remained localized in skin at the application siteavoiding unnecessary biodisposition to other organs with pro-spective minimization of toxic effects.

In recent years, researchers have compared NLCs to the tradi-tional SLNs and other lipid-based delivery systems. In one suchstudy, dibucaine (DBC)-loaded SLNs and NLCs were prepared by thehigh-pressure homogenization technique.222 Although DBC-loadedNLCs exhibited higher encapsulation efficiency (90.54 ± 0.95%)compared to its SLN counterpart (76.58 ± 7.88%), both nanocarriersshowed comparative significant decrease in its intrinsic cytotoxiceffect of DBC compared to control (free DBC solution).222 In anotherstudy, SLNs and NLCs of sildenafil (for treatment of erectiledysfunction) were prepared using a modified high-shear homog-enization method. Both nanocarriers exhibited small particle size(180 and 100 nm, respectively) and high entrapment efficiency(96.7% and 97.5%, respectively).223 Furthermore, permeation studyacross stratum corneum exhibited higher initial release from bothSLN and NLC formulations followed by controlled release, sug-gesting promising implications for faster onset and longer durationof action. It is worth noting that although both SLNs and NLCsdemonstrated almost similar initial permeation profile, however,after 5 h NLCs achieved higher skin permeation compared toSLNs.223 Similarly, other researchers have also compared SLN- andNLC-loaded clotrimazole and psoralen as well.31,224

Although NLCs overcome some of the major limitations of SLNs,however, lack of long-term stability data and regulatory challengeassociated with lipid particulate systems also impedes the successof NLCs in skin application area (Table 1).

Special Interest Areas

Transcutaneous Immunization

Vaccination triggers specific immune response and induceslong-lasting immunologic memory to protect against subsequentinfections. Almost all vaccines are administered by intramuscular(IM) or SC injection currently, which could be painful and requiresaseptic technique and trained personnel.225 Consequently, thistechnique is associated with poor patient compliance especially inchildren. In this regard, TCI provides a feasible cost-effectivealternative to the invasive routes of administration (IM and SC),along with providing uniform blood levels, reduced systemic sideeffects, and increased compliance.

Due to the presence of abundant LCs (dermal dendritic cells)beneath the epidermis, potent immune response could be seenwith TCI. The epidermal antigen presenting cells and migratoryT-lymphocytes are also present along with LCs. In fact, skin tissuecontains more antigen presenting cells than muscles and SC tis-sue.226 All these cells are collectively known as skin-associatedlymphoid tissue (SALT), which constitutes for the skin immunesystem. Cellular and humoral-mediated immune response can beseen because of SALT in association with lymph nodes.225,226

However, impervious nature of stratum corneum poses a signifi-cant challenge to deliver vaccine via skin route. The advances inelastic liposomes (especially ethosomes and ultradeformable li-posomes) for other disease areas have provided a unique oppor-tunity for TCI. Promising results for TCI has been recently reported

S. Jain et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-23 17

for elastic liposomes,227-230 SLNs,231 and nanoemulsion andmicroemulsion.232,233

In a recent study, flow cytometry and spectral bioimaginganalysis has provided clear evidence that Hepatitis B surface anti-geneloaded ethosomes were efficiently internalized by the den-dritic cells and were able to initiate an immune responsepredominantly of TH1 type.234 In another study, compared tointramuscularly administered alum-adsorbed PfMSP-119 solutionand topically applied PfMSP-119-loaded conventional liposomes,transcutaneous elastic liposomes improved immunization ofcarboxyl-terminal 19 kDa fragment of merozoite surface protein-1(PfMSP-119) of Plasmodium falciparum and induced robust andperdurable IgG-specific antibody and cytophilic isotype re-sponses.225 Similarly, transcutaneous immuno-boosts with adeformable vesicle Ag 85a (immuno-dominant antigens of Myco-bacterium tuberculosis which causes tuberculosis) combination inmice generated serum IgA and IgG2a, indicative of an IgA facili-tated, Th1-mediated immune response.235

In an interesting study, pH-sensitive diphtheria toxoid-loadedelastic vesicles were prepared using sucrose laurate ester,octaoxyethylene laurate ester and, sodium bis(tridecyl) sulfo-succinate with molar ratio of 5:5:1. It was observed that vesiclesexhibited antigen preserving potential and released antigen atdesired pH.236 Effect of loading of antigen in the deformable lipo-somes has also been studied on the particle size and entrapmentefficiency. Deformable liposomes loaded with Hepatitis B surfaceantigen plasmid DNA-cationic complex with lipid composition ofphosphatidylcholine, octadecylamine, and cholesterol in a weightratio of 9:1:1 resulted in particle size of 200 nm and entrapmentefficiency of 65%, with no effect of processing condition on the DNAintegrity. Furthermore, comparable to IM injection, similar sys-temic IgG response was obtained for the deformable liposomes.237

Although colloidal lipid-based delivery systems have providednew alternative for TCI, however, most of studies related to TCI areconducted in small animals. SALT, which constitutes for skin im-mune system, is significantly different from many small laboratoryanimal models. As a result, extrapolation of results from animalstudies to humans may not be well correlated.

Gene Delivery

There are 2 basic approaches for therapeutic gene delivery viaskin routedex vivo and in vivo gene delivery. Ex vivo delivery in-volves a skin biopsy to harvest cells externally for growth and geneinsertion in the culture, which is followed by regrafting to theskin.238 On the other hand, in vivo gene delivery involves directtransfer of the genetic material to the patient’s intact skin tissueand is therefore generally a more simple and direct approach.Compared to the viral vectors, nonviral vectors are considered safeand are therefore preferred.238 In this regard, topical application ofplasmid DNA is an attractive alternative approach for gene delivery.

Various lipid-based delivery systems have been utilized for genedelivery. A DOTAP-based SPACE (Skin Permeating And CellEntering) ethosomal system significantly enhanced siRNA in vitroskin penetration and accumulation to approximately 6 times and 10times, respectively, in porcine skin compared to the control(aqueous solution).239 In another study, physical stability evalua-tion of pMEL34-loaded elastic cationic niosomes revealed no visualsedimentation with only a slight increase in vesicular size and adecrease in zeta potential compared to initial when stored at 4�C,27�C, and 45�C for 8 weeks. Furthermore, the elastic cationicniosomes showed the highest tyrosinase gene expression in mel-anoma cell lines, indicating better tyrosinase activity compared tothe free and the loaded plasmid in nonelastic niosomes.158 Theeffect of type of edge activators on the transfection efficiency of the

plasmid DNA-loaded ultradeformable liposomes was also studied.It was found that bile salt-based edge activators (sodium cholateand sodium deoxycholate) are more suitable for transdermalabsorption of these DNA containing liposomes.108 Other promisingresults for elastic liposomes are also reported.240,241

Cationic lipid nanoparticles have also shown potential to effec-tively transfer plasmid DNA to the skin mainly due to the charge-mediated interaction between the complexes and the skin.242

Recently, N,N-bis(dimethylalkyl)-a,u-alkanediammonium surfac-tants, also called gemini surfactants, have received major attentionfor nonviral delivery systems. These surfactants have the ability tocondense plasmid DNA and self-assemble into small particles witha net positive charge that electrostatically interacts with anioniccell surface.243 Lipid nanoparticles constructed from tdTomatoplasmid DNA-cationic gemini surfactant, and a neutral lipid (DOPE)showed higher transfection efficiency in PAM-212 (Protospaceradjacent motif) cell line, compared to the control (commercialtransfection reagent Lipofectamine® Plus).243

Cosmeceuticals

Lipid-based delivery systems provide a unique asset forcosmetic industry. Table 4 represents the currently marketed lipid-based cosmetic products. Lipid in these delivery systems acts as anessential raw material for the regeneration of skin by replenishinglipid molecules and moisture. In most cases, this is enough toimprove skin elasticity and barrier function, which are the maincauses of skin aging. The lipid-based delivery systems utilizephysiological and biodegradable lipids that exhibit low toxicity, lowcytotoxicity, and better tolerability. Type of lipid in lipid-baseddelivery systems can affect the hydration efficiency on the skinsurface. In a study, egg phospholipids showed significantly higherhydration effects than soya phospholipids.244

Both conventional and elastic liposomes have shown promisingresults in the cosmeticeutical industry.245,246 Compared to freeresveratrol (a polyphenol with excellent antioxidant and free-radical scavenging properties), the liposomal formulation signifi-cantly enhanced the efficacy of resveratrol for treatment of UVradiation exposure.247 Furthermore, liposomal formulation pre-vented the cytotoxicity of resveratrol at high concentrations, evenat 100 M, by avoiding its immediate and massive intracellular dis-tribution.247 In another study, apigenin usage for treatment of skininflammation (induced by free radicals generated by UV, X-ray, orradiation) was studied using ethosomal delivery system.137 It wasobserved that apigenin-loaded ethosomes exhibited the strongesteffect on reducing cyclooxygenase-2 levels in mouse skin inflam-mation (induced by UVB light) compared to control (apigenin innormal saline). Synergistic effect of antioxidants, namely a-tocopherol and curcumin, was studied using niosomal deliverysystem. It was observed that coencapsulation of a-tocopherol andcurcumin in the vesicles exhibited 100% radical inhibition.248

However, more relevant work with regard to cosmetics is pub-lished for SLNs and LNCs. Lipid nanoparticles have exhibitedimproved chemical stability of cosmeceutical compounds that aresensitive to light, oxidation, and hydrolysis. The products NanoRepair Q10 cream and Nano Repair Q10 Serum (Dr. Kurt RichterLaboratorien GmbH, Berlin, Germany) introduced to the cosmeticmarket in October 2005 revealed the success of lipid nanoparticlesin the antiageing field. Enhancement of chemical stability afterincorporation into the lipid nanocarriers was proven for manycosmetic actives, for example, coenzyme Q10,249 ascorbyl palmi-tate,250 tocopherol (vitamin E), and retinol (vitamin A).251 Stearylferulate-based SLNs were found to improve the photostability ofantioxidant b-carotene and a-tocopherol to protect the skin againstUVA induced damages.199

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For Tretinoin (an antipsoriatic drug), lipid particulate systems(SLNs and NLCs) offered better photostability, skin transport, andantipsoriatic activity compared to liposomes, ethosomes, and themarketed product.252 In another study, the Q10-loaded NLCsexhibit a biphasic release pattern. NLCs provided a fast initialrelease followed by a slow release, while the Q10-loaded NEshowed a constant release over time.253 Furthermore, coencapsu-lating of antioxidant (luteolin) with sunscreen (octocrylene) inSLNs resulted in a higher photoprotection capacity compared to thecontrols.254 In an interesting study, even though SLNs (composed ofglyceryl dibehenate) and NLCs (composed of glyceryl dibehenateand caprylic capric triglyceride) exhibited similar occlusive effect,the penetration of a marker (Nile red) into the stratum corneumwas quite higher for NLCs than for SLNs. The results were attributedto the differences in the lipid composition of both these particulatesystems.255

A prolonged release is of interest for the perfumes as well as forthe perfumes that are incorporated into cosmetic products. It wasobserved that NLCs prolonged the release of the perfume Kenzocompared to emulsion and conventional shampoo.256 Furthermore,it was found that the release of perfume depends on the lipidmatrix composition, the perfume load, and the type of surfac-tant.201 Also, lipid nanoparticles have been successfully utilized todeliver diethyltoluamide, an ingredient of insect repellent products,by improving skin accumulation of the active and reducing its skinpermeation, which is essential for skin application of insectrepellents.257

Regulatory Aspect of Lipid-Based Delivery Systems

In general, the application of nanotechnology may result incertain product attributes that may differ from conventionallymanufactured products, and therefore may require specialconsiderationwith regard to safety and efficacy. In August 2012, theFDA’s Center for Drug Evaluation and Research provided an updateon its ongoing work related to the use of nanotechnology in drugproducts to the Advisory Committee for Pharmaceutical Scienceand Clinical Pharmacology.258 The update indicates that FDA hasevaluated its existing regulatory review processes for drug prod-ucts and determined that current procedures are adequate toidentify and address the potential risks associated with the use ofnanomaterials in drug products. However, some areas ofimprovement were also identified, especially the need for increasein regulatory science research and the training of review staff. TheFDA has also established specific policies and procedures forguiding internal reviewers of human drug and animal drug sub-missions. The FDA encourages industry to consult early with theagency to address questions related to the safety, effectiveness, orregulatory status of nanotechnology.258 These early consultationsafford an opportunity to clarify the manufacturer’s obligations anddiscuss methodologies and data needed to meet those obligations.

In 2014, the FDA issued a guidance for industry, entitled“Considering Whether an FDA Regulated Product Involves theApplication of Nanotechnology” to present its current thinking onnanotechnology and to seek public comment.259 As noted in thedraft guidance, the FDA has not adopted a regulatory definition ofnanotechnology or related terms. Based on this guidance, the FDAsuggests to consider 2 major points in determining whether an FDAregulated product involves the use of nanotechnology:

1. Whether a material or end product is engineered to have at leastone external dimension, or an internal or surface structure, inthe nanoscale range (approximately 1-100 nm).

2. Whether a material or end product is engineered to exhibitproperties or phenomena, including physical or chemical

properties or biological effects, those are attributable to its di-mension(s), even if these dimensions fall outside the nanoscalerange, up to 1 mm (1000 nm).

This suggests that in most cases, lipid vesicles and particulatesystems may come under the ambit of regulatory perspective for“nanotechnology.” Currently, there is no general guidance for lipidvesicles. However, the FDA issued draft guidance on October 29,2015 addressing the agency’s requirements for the submission ofnew drug applications, abbreviated new drug applications, or bio-logic license applications for liposome drug products.260 The newdraft guidance replaces Draft Guidance for Industry, Liposome DrugProducts, Chemistry, Manufacturing, and Controls; Human Pharma-cokinetics and Bioavailability; and Labeling Documentation publishedin August 2002. The guidance provides detailed recommendationsregarding physiochemical properties, description of manufacturingprocess, process controls, and control of excipients and drugproduct for new liposomal products. However, no recommenda-tions are provided for clinical efficacy and safety studies, nonclin-ical pharmacology/toxicology studies, or drug-lipid complexes.

In the guidance, the FDA recommends to provide the followingphysicochemical properties of drug product:

1. Morphology of the liposome, including lamellarity determi-nation, if applicable

2. Surface characteristics, as applicable3. Liposome structure and integrity, which refers to the ability

of the liposome drug formulation to contain the desired drugsubstance and to retain the drug substance inside theliposome

4. Zeta potential of the liposomes5. Viscosity of the drug product6. Drug encapsulation efficiency and liposome drug loading7. Particle size (i.e., mean and distribution profile)8. Phase transition temperature9. In vitro release of the drug substance from the liposome drug

product under the stated/described experimental conditionswith supportive data and information regarding the choice ofthose conditions Leakage rate of drug from the liposomesthroughout shelf life

10. Liposome integrity changes (e.g., release, containment effi-ciency, size) in response to changes in salt concentration

11. Spectroscopic data to support the proposed liposome struc-ture (e.g., phosphorus nuclear magnetic resonance)

Based on the evaluation of aforementioned physicochemicalproperties, critical quality attributes of the liposomal drug productshould be derived.261,262 Control of excipients should include allparameters which are necessary to define lipid components,including description, characterization, manufacture, and stability.Furthermore, lipid excipients fromnatural origin can have lot-to-lotvariability and requires proper control checks.

With regard to control of drug products, the recommendationsof the International Conference on Harmonization guideline Q6A“Specifications, Test Procedures and Acceptance criteria for NewDrug Substances and New Drug Products: Chemical Substances”are appropriate, but additional testing may be required. Consid-ering liposome drug products are sensitive to changes in themanufacturing conditions, in particular scale-up, it is important toestablish process controls to ensure liposome drug product quality.Prior knowledge and risk assessment of unit operation may beutilized to achieve efficient process control.

The guidance also emphasizes the need to provide adequatestability data to address the microbiological, physical, and chemicalstability of the liposome drug product, including the integrity of the

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liposomes in the drug product. Furthermore, studies for pharma-cokinetics and bioavailability are also recommended includingmass balance studies and pharmacokinetic studies.

Although the above-discussed information (along with someother available FDA literature) can provide some guideline fordevelopment of lipid-based delivery systems, there is no specificguideline in this regard for skin delivery. Lack of clear regulatoryguideline can pose a challenge in the development of these lipid-based delivery systems for commercial purpose.

Summary

Skin delivery provides an alternative to oral route, especially fordisease conditions that require targeted delivery. The major limi-tation for skin delivery is the impervious stratum corneum, whichlimits the entry of the drug through the skin. However, in the last 4decades, scientific and technological breakthroughs have led to thedevelopment of various lipid-based delivery systems for skin de-livery. Liposomes, ultradeformable liposomes, ethosomes, and lipidnanoparticles have shown successful delivery for variety of drugs.Despite the success of lipid-based delivery systems, stability issues,scalable manufacturing process, regulatory challenges, and cost aresome of the obstacles that require special consideration for suc-cessful delivery.

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