Nanoparticles and biomedicine

Department of Molecular Biochemistry and Pharmacology

AnastasiaBasta

Nano-World

• Synthesis of nanoparticles• In Vitro analysis - Nanotoxicity

• In vivo analysis

Milano, 1st March to 31th May 2014

AnastasiaBasta

1965 1973 1976 1989 1990 1992 1995 1996 1999 2004 2005 2009

Discover of liposomes

First liposomal drug formulation

First polymer-based nanoparticle

Long circulating pegylated liposomes

reported

First Liposome formulation enters

market

Polymer-based NPs enters clinical trials

NPs capable of crossing BBB

reported

First liposomal formulation approval

by FDA

Firts biologially active polymer approved by

FDA

Controlled-release polymer for brain

cancer apapproved by FDA

Polymer anticancer conjugate enters

clinical trials

First polymer-based micelle formulation of Doxo tranlated to

clinic

First protein-based NP approved by FDA

First targeted delivery of siRNA in humans

Petros and DeSimone, Nature Reviews, 2010

Early history of nanoparticles' therapeutics

Synthesis of NPs

Structure ofLiposomes

Gaucher disease is a progressive lysosomal storage disorder (LSD) caused by deficiancy of glucocerebrosidase leading to the dysfunction in multiple organ systems. Associated clinical symptoms include hepatosplenomegaly, anaemia, thrombocytopenia and skeletal deterioration.

Glucocerebrosidase enzyme replacement therapy has become the standard of care for patients with symptomatic Gaucher disease due to its safety and efficacy profile. The problem here is that the enzyme administrated cannot reach the blood-brain barrier, but remains at the peripheral organs. However, the BBB is permeable to small and lipophilic molecules.

Liposomes are colloidal vesicles ranging from few nanometers to several micrometers in diameter with one or more lipid layers. They are prepared from natural or synthetic phospholipids and cholesterol. In addition, they are biodegradable, biocompatible, non-toxic and non-immunogenic. They can, also, entrap a wide variety of therapeutic drugs, such us hydrophilic drugs. Liposomes are able to cross the BBB when they are connected with specific peptides.

The objective of the present study is therefore to exploit nanotechnology to use functionalised liposomes in order to deliver glucocerebrosidase to the central nervous system. The final goal will be the evaluation of the enzyme replacement therapy as possible beneficial effect in a murine model of LSDs.

Experimental procedure

Experimental plan For the preparation of liposomes, the dehydration-rehydration vesicles (DRV) method has been chosen.

Four different compositions have been tried:

I. Dioleoyl-Phosphocholine : Cholesterol : dipalmitoyl phosphoethanolamine-PEG-2000 (molar ratio 1 : 1 : 0,1)Lipids : Enzyme in a molar ratio of 90 : 2

II. Dioleoyl-Phosphocholine : Cholesterol : dipalmitoyl phosphoethanolamine-PEG-2000 (molar ratio 1 : 1 : 0,1)Lipids : Enzyme in a molar ratio of 90 : 2 Addition of sucrose during the rehydration phase (w/w 1:1 sucrose to lipids)

III. Phosphatidylcholine : Cholesterol : Phosphatidic acid (molar ratio 7 : 2 : 1) Lipids : Enzyme in a molar ratio of 90 : 2

IV. Phosphatidylcholine : Cholesterol : Phosphatidic acid (molar ratio 7 : 2 : 1)Lipids : Enzyme in a molar ratio of 90 : 2 Addition of sucrose during the rehydration phase (w/w 1:1 sucrose to lipids)

(chloroform)

in evaporator

with PBS

with PBS(multilamellar

vesicle s)

184000 g

Supernatant

Through Bradford analysis we quantified the µg of enzyme entrapped in liposome and free in the supernatant. Inside liposomes we found about 38µg, while in the supernatant 810 µg. They correspond respectively to 3% and to 60 % of the initial amount of the added enzyme (1337μg) . These values remained stable also 24h after the synthesis.

Then we performed the measurement of enzymatic activity by using a fixed concentration of the substrate (3 mM) and a fixed incubation time (30 minutes). We correlated the fluorescence intensity obtained from the reaction with the concentration of the active enzyme in each sample. By matching these values we obtained the values of enzymatic activity expressed as units (µmol substrate transformed/min/mg protein).

Liposome contained 0.7 U, while in the supernatant there were 0.9 U of enzyme. Therefore, only 1,5% of the enzymatic activity is still measurable and remains stable also 24 hours after liposome preparation.The mixture containing free and entrapped enzyme corresponds to 1,6 U, i.e. to 50 µg of active enzyme.

µg of protein in 500 μl of sample In liposomes 38µg

In the supernatant 810µg

Total 848µg

μg of active protein in 500 μl of sample In liposome 21,8µg 0,7 U

In supernatant 28,4µg 0,9 U

total 50,2µg 1,6 U

Results - Discussion

Supernatant Supernatant

In Vitro analysis - Nanotoxicity Standard Operating Procedure with A549 cells

The A549 cell line is a human cell line originated from lung carcinomatous tissue. The cells have an epithelial morphology and are adherent.

Knowledge of the relative growth rate of a specific cell type is useful for planning experiments.

Seeding at 0.5x105 cells/well in cell medium, 8 replicates per time period (24h, 48h, 72h)

10x 10x10x

Control conditions

hours

Cel

ls/m

l

0 24 48 720

50000

100000

150000

200000

250000

300000

350000

0,05% (w/v) Trypsin-EDTA has been added to cultures 24h, 48h, 72h after seeding to detach cells from the seeding plate.

An automatic cell counter has been used to measure the amount of living cells.

According to the results shown the cells are healthy, their morphology is appropriate and their doubling time is about 30 hours.

Standard Operating Procedure MTS cell viability assay

The MTS tetrazolium (Owen’s reagent) is reduced by mitochondria of viable cells into a colored formazan derivative that is soluble in tissue culture medium. Assays are performed by adding a small amount of the reagent directly to culture wells. After 1h of incubation the absorbance at 490nm was determined.

Cells were seeded at 1x104 cells/well

1) Add MTS reagent 2) Incubate for 1h 3) Measure O.D at 490 nm

• NPs added in the cells culture: amine-modified polystyrene nanopartices, 50 nm diameter

carboxyl-modified polystyrene nanopartices, 40 nm diameter

• NP dilutions: 100 µg/ml, 50 µg/ml, 25 µg/ml, 10 µg/ml, 1 µg/ml

Steps of the assay

Nanotoxicity assay

Representative results following incubation with NPs

1µg/ml 10µg/ml

25µg/ml 50µg/ml

100µg/ml

POSITIVE CHARGED NPs

1µg/ml 10µg/ml

25µg/ml 50µg/ml

100µg/ml

NEGATIVE CHARGED NPs

Morphology, apparent health, percentage of confluence

200μm 200μm

Mycoplasma testTest for mycoplasma should be performed as a quality check of the cell cultures.

Ctr

+ D

NA

M

ycoM

arke

r

Ctr

-

(H2O

)M

EM

26

-3

ME

M 2

8-3 M

EM

31-

3 ME

M 4

-4 M

EM

7-

4 ME

M 8

-4 M

EM

9-

4 ME

M 1

1-4

ME

M 1

4-4

1.5Kb

1.0Kb

0.5Kb

0.7 Kb

expected amplified product at 700bp

Negative mycoplasma results have been obtained by electrophoresis analysis

In Vivo analysis

• Administration (iv) of 20 mg/kg of NPs.• Two mice sacrificed 1h, 4h, 24h and 48h after administration• Two kinds of NPs used:

Core Shell NPs and PolyCaproLactone 3 (PCL3) NPs• Tumor and liver sections (20 µm thick) were acquired with fluorescent microscope• Nuclei are stained with HOECHST (blue signal) and NPs are visualized in red through

Rhodamine B (red) signal.• Only tumor samples of mice sacrified 24h and 48h after iv injection underwent to

histological analysis.

Biodistribution of Core Shell NPs and PolyCaproLactone 3 (PCL3) NPs loaded with Paclitaxel (PTX) in a mouse model of triple negative breast cancer

Core Shell NPs PCL3 NPs

Core shell NPs in Liver

NPs are concentrated in the veins where it is possible to see an association between circulating cells and red staining. No relevant migration was observed in parenchyma

A large amount of signal is still confined to the small vessels and almost disappeared in larger vessels. However, a significant migration to the parenchyma is clearly detectable.

4h - Liver1h - Liver

A strong migration in liver parenchyma can be observed. No presence of NPs are found in smallest vessels (brighter nuclei forming a circular structure). This widespread distribution suggests the presence of a specific association between nuclei and NPs.

24h - Liver 48h - Liver

A high amount of NPs are found in the liver parenchyma but not inside the big vessels.

40x

Epi Fluorescence Microscopy 20x

100μm 100μm

100μm 100μm

50μm

Core shell NPs in Tumors

Here, the general pattern of distribution in tumor sections is confirmed: NPs are visible after diffusing in the parenchyma both as internalized in cells and as big clusters not specifically associated with cellular internalization.

24h - Tumor 48h - Tumor

In this picture a very strong signal can be clearly observed: NPs spread in the parenchyma as clusters or as specified signal associated with cells, but vessels are out of signal.

Epi Fluorescence Microscopy 20x

100μm 100μm

PCL3 NPs in Liver

1h - Liver

It is possible to see blue dots inside the vein. This means that circulating cells are associated with NPs at this time. No relevant spread of NPs was here observed in parenchyma.

4h - Liver

In this picture, it is possible to see a strong signal associated with NPs at the external layer of this big vessel. It is also noteworthy that a signal associated to the migration of NPs -from the vessel to the parenchyma around- is observed.

24h - Liver

NPs can be found on both small vessels walls (all around endothelial layer) and in the parenchyma.

48h - Liver

NPs seem to be almost homogenously localized in the parenchyma. Vessels, either big or small ones, seem to be completely empty.

Epi Fluorescence Microscopy 20x

100μm

100μm100μm

100μm

PCL3 NPs in Tumors

A highly heterogeneous staining is visible. In many cases NPs seem to be uptaken by cells

24h - Tumor

NPs are heterogeneously diffused in the parenchyma. Many coronal vessels are contoured by a thin layer of NPs that extravasated.

48h - Tumor

Epi Fluorescence Microscopy 20x

100μm100μm

Optimization of immunohistochemistry protocols in organs of mice treated with NPs

• Mice were treated with PCL3 and Core Shell NPs.

• We wanted to evaluate: - the extravasation of NPs from the blood vessels to the parenchyma (endothelium staining with CD31 antibody) - the accumulation in macrophages (staining with IBA1 antibody)

• To optimize the immunohistochemistry protocols we obtained liver sections of a mouse treated with PCL3 NPs and sacrificed 4 hours after injection

• Thirty μm thick sections were obtained with a criostat at -25°C and were put onto cover glass slides following a standardized protocol in order to obtain a serial reconstruction of the entire volume of the organ.

Ab anti-A

Ab anti-anti-A

Fluorescence/staining Tag

principle of immunohistochemistry

Protocols used for the treatment of tissues with antibodies

CD31 Ab IBA I Ab

Confocalmicroscopy

RESULTS & DISCUSSION

CD31 NPs can be found both in the blood vessels and homogenously distributed in the

parenchyma of the organ . Partial colocalization of NPs and endothelium

are also able to be observed.

20μm

1:50

20x Epifluorescence images of HOECHST (blue), CD31 (green) and RhB (red)

Confocal Microscopy images of HOECHST (blue), CD31 (green) and Rhodamine B (red)

40x 60x

1:100 1:100

60x50μm 20μm

100μm 100μm

100μm 100μm

50μm

IBA I

A strong signal associated to NPs was confirmed both in vessels and in organ parenchyma.

100μm

100μm 100μm 20μm

By checking the general biodistribution of NPs it was able to find that both Core Shell NPs and PCL3 NPs show a similar behaviour :

In the first hour after administration, NPs can be observed almost exclusively inside the vessels of the organs.

However, 4 hours later of the administration, NPs are located both in vessels and in the parenchyma of the tissue.

Finally, at the time point of the 24 hours and over, NPs can be found spread in the parenchyma both in liver parenchima and in the tumor.

Conclusions

Ending…

Future steps of nanomedicine….

Synthesis

In vitro

In vivo

I have a dreamWe can envision a future where tiny nanomaterials (nanodroids,

ISAC ASIMOV) travel through the bloodstream searching for sites of infection or disease processes, and once any irregular cellular activities are found, they trigger the release of nanomaterial cargoes that repair damaged tissues or kills foreign invaders. (self-healing strategy)

Many thanks for this experience and

educational period to:Prof. Mario Salmona

Dr. Paolo BiginiDr. Leopoldo Sitia

Dr. Martina Violatto*Biochemistry Lab