Layer-by-layer assembly of graphene oxide on thermosensitive liposomes for photo-chemotherapy

Mohadeseh Hashemia,b,c, Meisam Omidie,f, Bharadwaj Muralidharanb,d, Lobat Tayebie, Matthew J. Herpinc, Mohammad Ali Mohagheghig, Javad Mohammadia,*, Hugh D.C.

Smythc,*, Thomas E. Milnerb,*

a Department of Biomedical Engineering, Faculty of New Sciences and Technologies, The University of Tehran, Tehran, IranbDepartment of Biomedical Engineering, The University of Texas at Austin, Austin, USAc Division of Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, USAd Department of Electrical and Computer Engineering, The University of Texas at Austin, Austin, USAe Department of Developmental Sciences, Marquette University School of Dentistry, Milwaukee, USA f Protein Research Center, Shahid Beheshti University, GC, Velenjak, Tehran, Irang Cancer Research Center, Cancer Institute of Iran, Tehran University of Medical Sciences, Tehran, Iran

*E mail: Javad Mohammdi (hashemi@utexas.edu), Hugh D Smyth (hugh.smyth@austin.utexas.edu), Thomas E Milner (terra.laser@gmail.com)

Supplementary 1 - FTIR spectrum of GO, GO-PLL, and PLL

Figure S1. A) FTIR spectra of GO, GO-PLL, and B) PLL

FTIR analysis confirmed covalent grafting of GO with PLL molecules (Figure S1). The FTIR spectrum of GO confirmed the presence of C-O (1129 cm-1), C=C (1622 cm-

1), C=O (1733 cm-1), and O-H (3386 cm-1) bonds. The FTIR spectrum of GO-PLL showed characteristic peaks of PLL noted by the peaks of N-H (3271 cm-1) and C=O (1624 cm-1) bonds (Figure S1B). Moreover, the presence of C-N stretching bond in GO-PLL (1314 cm-1) proved the successfully conjugation of the amine group in PLL molecules to epoxy group of GO nanosheets.

Supplementary 2 - Evaluation of hydrodynamic size and zeta potential 

Figure S2. In Figure S2A, effect of ionic strength on the zeta potential of GO and GO-PLL is given. In Figure S2B, the effect of pH variation on protonation and deprotonation of GO (red line) and GO-PLL (black line) were monitored in 10 mmol.L-1 NaCl. The physiological stability of cationic liposomes was studied by monitoring the zeta potential (blue line) and size (right y-axis, green line) of the surface at different pH levels.

In Figure S2A, effect of ionic strength on the zeta potential of GO and GO-PLL is given. In Figure S2B, the effect of pH variation on protonation and deprotonation of GO and GO-PLL were monitored in 10 mmol.L-1 NaCl. The physiological stability of cationic liposomes was studied by monitoring the size and zeta potential of the surface at different pH levels.

Supplementary 3 - Estimating the number of GO sheets required to completely cover the surface of the liposomes

The number of GO sheets needed to cover the liposomal surface was predicted by estimating the number of GO nanosheets and liposomes in the suspensions. Surface area of both GO nanosheets and liposomes was then estimated using DLS analysis and TEM image (Figure 3). Theoretically, the number of liposomes in the suspension has been calculated based on the method proposed by Hila et al.[1] By assuming that the liposomes are unilamellar, the number of lipid molecule can be calculated by the equation below:

N tot=[4 π ( d

2 )2

+4 π [ d2−h]

2

]

a (S1)

Where d is the diameter of the liposome, h is the thickness of the bilayer (approximately 5 nm), and a is surface area of the lipid head group. As the main composition in the formulation is phosphatidylcholine, we substitute the surface area of the head group of phosphatidylcholines as approximately 0.71 nm2. In the above equation, the surface area of inner and outer sides of bilayers in a unilamellar liposome are added together, then the total lipid area was divided to the head group area of one lipid molecule.Number of liposomes per ml is then calculated by:

N Lipo=M lipid × N A

N tot ×1000 (S2)

Where N A is Avogadro’s number (6.022× 1023), M lipid is the molar concentration of the lipid and N tot is the total number of lipid molecules per liposome. Herein, the z average diameter of the liposomes are 138.8 nm and the molar

concentration of the liposome measured by a Stewart assay is 1.36 μmol

ml . Hence, the

number of liposome molecules in 500 µl of the suspension is 592.66×1010. Prior to calculating the number of GO sheets in the suspension, the molecular weight of GO is roughly estimated. A sheet of GO is structured with a hexagonal ring-based carbon network and hence, in 1 nm2 of GO, there are 46.5 carbon atoms. Based on the data sheets provided by Graphene supermarket, the GO sheets are composed of 30 % oxygen. Thus, the number of oxygen atoms in 1 nm2 of graphene oxide sheets is 13.95. Based on the specifications, the molecular weight of 1 nm2 of GO is calculated to be 764.4 mg/mmol, approximately (by neglecting the H group).The surface area of GO sheets was determined based on a TEM image (Figure 3) using Image J software (Windows version; National Institutes of Health). The average surface area is estimated at around 2×104 nm2. Based on the estimated molecular

weight for 1 nm2 of GO, the molecular weight of each sheet is around 110 kDa. By assuming that the conjugation reaction of PLL to the surface of GO was complete, the molecular weight of GO-PLL is computed at around 335 kDa.Regarding these estimates, the molar ratio of GO and liposome were calculated in Table 1. Furthermore, considering a TEM image of GO and DLS data of liposomes, the minimum molar ratio of GO/ liposome, for an effective coating of GO on the surface liposome, is 3 (see Table 1).

Also, the molar ratio of GO-PLL nanosheets was estimated, as explained previously, and represented in Table 1. Computing the amount of GO-PLL needed for second layer coating was infeasible due to the heterogeneous structure of 1 LBL Lipo-graph.

Supplementary 4 - The effect of GO concentration on 1 LBL Lipo-graph formation

Figure S3. The effect of GO/Liposome mass ratio (1/2, 1/4, 1/8, and 1/16) on 1 LBL Lipo-graph construction

As discussed in Supplement 3, for mass ratio of GO/Liposome=1/4, the molar ratio of GO and liposome are equal which leads to the formation of large fractal aggregates.

Supplementary 5 - UV-Vis spectra of GO and GO-PLL

Figure S4. UV-Vis absorption spectra of GO and GO-PLL

Supplementary 6 - Calculation of absorption cross section of 4 LBL Lipo-graph

The absorption cross-section of GO, GO-PLL and 4 LBL Lipo-graph was obtained based on the experimental setup described by Hashemi et al. [2].

6.1. Minimization of scattering and lateral thermal diffusion

To minimize lateral thermal diffusion, the lateral thermal relaxation time is calculated as:

τ= d2

16 x (S3)

where,x is the thermal diffusivity of water (0.14 mm2.s-1) and d is the laser beam diameter (2 mm). Based on equation S3, lateral thermal relaxation time is 1.78 s. Hence, a laser pulse with repetition rate of 2 Hz (50% duty cycle) is chosen, so the pulse duration (0.25s) is well below the lateral thermal relaxation time.To reduce the effect of light scattering on the computation, an appropriate concentration of GO, GO-PLL and 4 LBL Lipo-graph is experimentally determined, by comparing the temperature profile of various GO concentrations (0.25, 0.125, and 0.0625 mg.ml-1 ), GO-PLL (0.1, 0.15, and 0.2 mg.ml-1 ), and 4 LBL Lipo-graph ( 0.1, 0.2, and 0.3 mg.ml-1 ) with the temperature profile of standard black absorber (Figure S5 A-D). The maximum concentration having a similar temperature profile spread to the standard black absorber is chosen as a concentration with minimal light scattering effect. The optimized concentration of GO, GO-PLL and 4 LBL Lipo-graph that met the afore-mentioned condition, are 0.25, 0.2 and 0.3 mg.ml-1, respectively.

6.2. Absorption Cross section of GO, GO-PLL, 4 LBL Lipo-graph

By reducing the effect of light scattering and lateral thermal diffusion, the infrared emission detected during pulsed laser irradiation is predominantly directly absorbed by the nanoparticles. The heat absorbed by the nanoparticle is given by

dQdt

= mC dTdt (S4)

where Q is the heat generated during laser emission [ J ], m is the mass of the

nanoparticle [ kg ], C is the specific heat capacity [Jkg.K ] .By substituting

dQdtV

= μa ×φ and mV

= ρ in the equation above:

dT(t)dt

=μa ×φ(t)ρC

(S5)

where μa is the absorption coefficient [ L⋅mol-1 ⋅cm-1 ], φ is laser beam fluency [Wm2 ], ρ

is material density [kgm3 ]. By substituting analytic signals [2] for laser fluence rate and

temperature φ=φ0e i2πft and T=T0 ei2πft in equation above, the magnitude of absorption coefficient is given by:

μa =2πfρC T0

φo (S6)

So, the absorption cross section of the nanoparticles is calculated by using:

µa =σ*C (S7)

where σ is absorption cross section [mlmg.m ], and C is the corresponding concentration [

mgml

¿.

Temperature per pulse,T 0, is calculated by placing 400 µl of the nanoparticles in a 96 well-plate, irradiating with a 808 nm NIR laser pulse (2 Hz at 50 % duty cycle) and monitoring radiometric temperature rise for sixty seconds (Figure S5 E-G). Then the measured temporal profile is filtered with a band pass filter centered at 2 Hz, to make sure that the slope is not affected by intensity noise (Figure S5 H-J). The steady state temperature per pulse for GO, GO-PLL and 4 LBL Lipo-graph respectively is 0.16 ± 0.01, 0.00675 ± 0.00075, and 0.015 ± 0.001 .℃

By substituting φ0=1.77 [ Wcm 2 ], ρ =1001.77 [kg

m3 ], C = 4178.62  [Jkg.K ], and f =2 Hz, the

absorption coefficient and absorption cross section of GO, GO-PLL and 4 LBL Lipo-graph was calculated. The results are tabulated in Table S1. The absorption cross section for GO is similar to the value that previously reported [3].

Table S1. Absorption coefficient and absorption cross section of GO, GO-PLL, 4 LBL Lipo-graph

Absorption coefficient (µa)[cm-1]

Absorption cross section (σ)

[ml .(mg.cm)-1 ]GO 4.7 ± 0.30 19 ± 1.2

GO-PLL 0.2 ± 0.022 1 ± 0.114 LBL Lipo-graph 0.44 ± 0.03 1.47 ± 0.1

Figure S5. Scattering profile of A) black absorber, B) GO, C) GO-PLL, D) 4 LBL Lipo-graph at various concentrations. Temporal profile of radiometric temperature versus time of E) GO, F) GO-PLL, and G) 4 LBL Lipo-graph. Filtering the data of H) plot E, I) plot F, and J) plot G

Additional reference

[1] H. Epstein, E. Afergan, T. Moise, Y. Richter, Y. Rudich, G. Golomb, Number-concentration of nanoparticles in liposomal and polymeric multiparticulate preparations: Empirical and calculation methods, Biomaterials 27(4) (2006) 651-659.[2] J.W. Goodman, Statistical optics, John Wiley & Sons (2015).[3] M. Hashemi, M. Omidi, B. Muralidharan, H.D. Smyth, M.A. Mohagheghi, J. Mohammadi, T.E. Milner, Evaluation of the Photothermal Properties of a Reduced Graphene Oxide/Arginine Nanostructure for Near-Infrared Absorption, ACS Applied Materials & Interfaces 9 (38) (2017) 32607–32620.