Supplementary Information

Co-delivery of docetaxel and gemcitabine by anacardic acid modified self-assembled

albumin nanoparticles for effective breast cancer management

Varun Kushwah1,2,3, Sameer S. Katiyar1, Chander Parkash Dora1, Ashish Kumar Agrawal2,

Dimitrios A. Lamprou 3,4, Ramesh C. Gupta2, Sanyog Jain1*

1Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of

Pharmaceutical Education and Research, SAS Nagar, Punjab, India.

2James Graham Brown Cancer Centre, University of Louisville, Louisville, KY, USA.

3Strathclyde Institute of Pharmacy & Biomedical Sciences (SIPBS), University of Strathclyde,

Cathedral Street, Glasgow, G4 0RE, United Kingdom.

4Medway School of Pharmacy, University of Kent, Medway Campus, Anson Building, Central

Avenue, Chatham Maritime, Chatham, Kent, ME4 4TB, United Kingdom.

* To whom correspondence should be addressed: E-mail: sanyogjain@niper.ac.in,

sanyogjain@rediffmail.com, Tel.: +91-172-2292055, Fax: +91-172-2214692

Supplementary Material

1 METHODS

1.1 Method

1.1.1 Analytical method development and validation for GEM

HPLC method was developed and validated for the quantitative determination of GEM as per

ICH guidelines. The HPLC system was comprised of a Waters 2695 separation module equipped

with a quaternary pump, an auto sampler unit, and a Waters 2996 photodiode array (PDA)

detector equipped with Empower software. Stock solution of GEM (100 µg/ml) was prepared by

dissolving 5 mg in 50 ml of water (HPLC grade) and diluted in appropriate concentrations as

working standard solutions. Chromatographic separations were done using the reversed phase

C18 column (250 mm × 4.6 mm, 5μm; Thermo Scientific, USA) with parameters listed in Table

S1.

Table S1: HPLC method parameters for in-vitro estimation of GEM

Parameters ValuesMobile Phase Phosphate buffer (pH 3.5, 0.05M): methanol (40:60)Column RP- C18 (250 mm × 4.6 mm. 5 μm; Thermo Scientific, USA)Elution IsocraticFlow Rate 0.8 ml/minRetention Time 5.3 minRun Time 11.0 minColumn Temp. 25 °Clmax 268 nmInjection Volume 20 mlDetector PDALC Software Shimadzu, LC solution1.1.2 Bioanalytical method development for GEM

For quantitative determination of GEM in plasma, bioanalytical method was developed with

HPLC. Briefly, 20 μl of tetrahydrouridine (1mg/ml) was added to 100 μl of plasma to inhibit

cytidine deaminase. Then, 25 μl of an aqueous solution of drug and internal standard (IS; 2’-

deoxycytidine, 10 μg/ml) was added to 100 μl of plasma and the sample was vortex mixed. 50 μl

of 20% w/v trichloroacetic acid was added and the mixture was thoroughly vortex mixed prior to

centrifugation at 10,000 rpm for 5 min. The supernatant was transferred into a vial. Again, 50 μl

of 20 % trichloroacetic acid was added to the precipitate and, after brief vortex mixing,

centrifugation was repeated. The combined supernatant was collected with 100 μl of mobile

phase and transferred to an auto-sampler vial and analyzed. Table S2 lists various HPLC

parameters used for bioanalytical method development.

Table S2: HPLC method parameters for bioanalytical estimation of GEM

Parameter ValueMobile Phase Acetate buffer (20mM, pH 5.0): ACN (97: 3)Column RP- C18 (250 mm × 4.6 mm. 5μm; Thermo Scientific, USA)Elution IsocraticFlow Rate 1 ml/minRetention Time GEM: 8.17 min, IS: 12.97 minRun Time 20.0 minlmax GEM and IS; 272 nmInjection Volume 60 mlDetector PDALC Software Shimadzu, LC solution1.1.3 Analytical method development and validation for DTX

Analytical method for docetaxel was developed and validated. Detection wavelength for DTX

was found to be 230 nm and rest parameters are listed in Table S3.

Table S3: Chromatographic conditions for HPLC analytical methodParameter In-vitro methodMobile phase ACN: Ortho Phosphoric acid Buffer (53:47% v/v)Column C18 5 µm (MACHERE-NAGEL)Elution Isocratic elutionFlow rate 1 mL/minRun time 10 minutesColumn temperature 35ºCWavelength 230 nmInjection volume 20 µLDetector PDALC Software Shimadzu, LC solution

1.1.4 Bioanalytical method development for DTX

Plasma concentrations of DTX were determined by validated HPLC assay. Calibration curve

were used for the conversion of DTX/PTX chromatographic area to the concentration of DTX.

For the calibration curve firstly, 25 µl of blood plasma was taken in an eppendorf followed by

addition of 5 µl paclitaxel solution (as an internal standard for both DTX) from previously

prepared 1 mg/ml of paclitaxel solution in methanol. From the stock of DTX (1 mg/ml) further

samples were prepared for calibration curve in the range from 10 ng/ml to 1µg/ml of DTX. Later

samples were diluted upto 1 ml with acetonitrile: methanol (50:50) mixture for protein

precipitation and vortexed for 15 sec. The mixture was centrifuged for 10 min at 10000 rpm.

After that supernatant was taken and dried in vacuum oven. Post drying samples were

redispersed in 300 µl of acetonitrile: methanol (50:50) mixture and again centrifuged for 10 min

at 8000 rpm. Finally, supernatants were collected and injected in HPLC.

Table S4: HPLC method parameters for bioanalytical estimation of DTXParameter In-vitro methodMobile phase ACN: Ortho Phosphoric acid Buffer (53:47% v/v)Column C18 5 µm (MACHERE-NAGEL)Elution Isocratic elutionFlow rate 1 mL/minRun time 12 minutesColumn temperature 35ºCWavelength 230 nmInjection volume 60 µLDetector PDALC Software Shimadzu, LC solution

1.2 Synthesis of anacardic acid–gemcitabine-bovine serum albumin dual drug conjugate

(AA-GEM-BSA)

AA-GEM-BSA dual drug conjugate was synthesized utilizing free -COOH and –NH2 groups of

BSA which binds with Gemcitabine (GEM) and Anacardic Acid (AA), respectively. The

covalent modification of these groups involved three sequential steps: (i) EDC and NHS

activation of –COOH of AA (ii) Covalent conjugation with free –NH2 group of BSA with

activated AA; (iii) EDC and NHS activation of free –COOH of BSA (iv) Conjugation of

activated –COOH of BSA with free –NH2 group of Gemcitabine (GEM).

Briefly, AA (10 mg) dissolved in ethanol (20 ml) in round bottom flask and EDC (5.6 mg) and

NHS (3.38 mg) dissolved in ethanol (1 ml) was added and allowed to stir for 12 h at room

temperature. The reaction was performed in presence of anhydrous triethylamine as base. Then

100 mg BSA dissolved in water (20 ml) was added and allowed to stir for 24 h at room

temperature as a result activated carboxylic group was conjugated with free amine group of

BSA. Furthermore, for the activation of – COOH group of BSA, EDC (6.4 mg) and NHS (3.86

mg) dissolved in ethanol (1 ml) was added in the reaction mixture and allowed to stir for 12 h at

room temperature. To the resultant mixture, GEM (10 mg) in water (1 ml) were added and

allowed to stir at 350 rpm for 24 h at room temperature. The prepared conjugate was precipitated

at pH 5 and separated by centrifugation at 21000 rpm for 10 min. Purification of the conjugate

was performed by repeated washings with water and lyophilized. Schematic representation of

Docetaxel-Gemcitabine direct conjugate synthesis is given in Synthesis Scheme S 1.

1.3 Spectroscopic evaluation

Synthesized conjugates were subjected to exhaustive evaluation using FTIR, 1H NMR, UV

Visible spectroscopy.

State of aggregation: Whitish brown; Yield: 81.4%

UV vis (λmax, nm): 229, 278

1H NMR of AA (δ, DMSO-d6, ppm, 400 MHz): 7.14 (1H, t, H-4); 6.70 (1H, d, H-3); 6.66 (1H,

d, H-5); 5.32 (2H, m, H-8’ and H-9’); 2.58 (2H, t, H-1’); 1.99-1.98 (4H, m, H-10’ and H-7’);

1.49 (2H, p, H-2’); 1.30-1.21 (16H, m, H-3’, H-4’, H-5’, H-6’, H-11’, H-12’, H-13’ and H-14’);

0.86 (3H, t, H-15’).

1H NMR of GEM (δ, DMSO-d6, ppm, 400 MHz): 9.89 (1H, s, H-1’’); 8.79 (1H, s, H-1’’);

8.14 (1H, d, H-2’’); 6.43 (1H, s, H-3’’); 6.22 (1H, d, H-4’’); 6.08 (1H, t, H-5’’); 4.23-4.15 (1H,

m, H-6’’); 3.92-3.90 (1H, m, H-7’’); 3.80-3.77 (1H, dd, H-8’’); 3.66-3.62 (1H, dd, H-8’’).

1H NMR of AA–BSA-GEM conjugate (δ, DMSO-d6, ppm, 400 MHz): 7.96-6.35 (m,

aromatic protons of BSA and AA); 5.32 (2H, m, H-8’ and H-9’); 4.37-3.74 (4H, m, H-6’’, H-7’’

and H-8’’); 2.59 (2H, t, H-1’); 2.00-1.98 (4H, m, H-10’ and H-7’); 1.45-0.86 (m, aliphatic

protons of BSA and AA); 0.85 (3H, t, H-15’). (Figure S 1).

FTIR (νmax, KBr pellets cm-1): 3600-3200 (O-H, N-H), 3200-2850 (aromatic and aliphatic C-H),

1660 (CO of amide bond), 1539 (bending for secondary amide), 1450 and 1375 (-CH3)1400-

1000 (C-F), 1350-1000 (C-N, C-O), 1229 (C-O-C), 1038 (C-C), 900-690 (bending vibrations of

aromatic C-H) (Figure S 2).

O

N

O

O OH

OH

EDC, NHS

O O

OH

O NH -BSA-COOH

OH

EDC, NHS

NH2 -ALB-COOH

O

OH

O

N

O

N

ON

NH2

OHO

HO FF

N

ON

HN

O OH

OHF F

O

OH

NH -BSA-CO

NH -BSA-CO

Synthesis Scheme S 1: A schematic representation depicting the synthesis of AA–BSA-GEM

conjugate

H2O

1’’ 1’’ 2’’ 4’’

5’’ 6’’ 7’’ 8’’ 8’’

DMSO

3’’

N

ON

NH2

OHO

HO FF

1''

2'' 4''3''

5''6''

7''8''

A

B

C

O OH

OH123

456

1'

2'

3'

4'

5'

6'

7'

8'

9'

10'

11'

12'

13'

14'

15'

DMSO

4 3,5

8’,9’

1’ 7’,10’

2’

3’-6’ 11’-14’

15’

N

ON

HN

O OH

OHF F

NH -BSA-CO

OH123

456

1'

2'

3'

4'

5'

6'

7'

8'

9'

10'

11'

12'

13'

14'

15' 1'' 2''4'' 3''

5'' 6'' 7''

8''

O

4 3,5 8’,9’

Aliphatic protons of BSA

15’

7’,10’ 1’

6’’-8’’

Aromatic protons of BSA

Figure S 1: H1 NMR of A: AA; B: GEM; C: AA-GEM-BSA Conjugate.

AA-GEM-BSA

AA

GEM

BSA

Figure S 2: Overlay FTIR spectra of AA, GEM, BSA and AA-GEM-BSA conjugate.

1.4 Characterization of AA-GEM-BSA conjugate

1.4.1 Degree of modification (TNBS and titration method)

The extent of conjugation between AA and BSA was measured by evaluating the free amino

groups in BSA via 2,4,6 trinitrobenzenesulfonic acid (TNBS) reagent (1, 2). Briefly, 200 µL of 1

mg/mL BSA (3.0303 µM final assay concentration) and AA-GEM-BSA conjugate solution was

added to 600 µL 0.1 M sodium bicarbonate buffer (pH 8.5). TNBS (200 µL, 0.1%) was added to

the above mixture and kept for incubation at 37 °C in dark for 2 h. The absorbance of each

solution was measured at 340 nm using UV/Vis spectrophotometers (BioTek, USA). The molar

concentration of amine groups present in each sample was measured by using standard

calibration curve of known L-lysine concentrations (Eq. 1)

DM=CBSA−CGEM−BSA /CBSA× 100 (1)

Where, CBSA and CGEM−BSA are the concentration of free primary amino in BSA and AA-GEM-

BSA conjugate, respectively.

While, the carboxylic group determination was preformed via titration method with

phenolphthalein as indicator. Briefly, a solution of 0.01 N NaOH in water was prepared and in a

separate conical flask a defined volume of the solution of BSA and conjugate was poured.

Further, few drops of phenolphthalein indicator was added and titrate with standardized NaOH

until a faint pink colour appears. Finally, the equivalent volume of NaOH was recorded and the

normality was calculated via equation

N1V1=N2V2 (2)

Where, N2 can be related to available COOH groups.

1.4.2 Fluorescence spectroscopy

The conjugation of AA and GEM with BSA was further analyzed by measuring the intrinsic

tryptophan fluorescence emission spectra of BSA, AA-BSA, GEM-BSA and AA-GEM-BSA

conjugate. Fluorescence readings of samples were excited at 280 and the emission was recorded

in the range from 300-400 (Peak fluorescence intensity at 360 nm) in 96 well plate fluorescence

reader Synergy 2, BioTek.

1.4.3 MALDI-TOF analysis

The molecular weight of native and AA-GEM modified BSA conjugate was determined by the

Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectroscopy

(Shimadzu, Axima-CFR spectrometer, mass range 1–150 000 Da).

1.4.4 SDS gel electrophoresis

The molecular weight of AA-GEM conjugated BSA and native BSA was further analyzed via

sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, different BSA

samples were loaded in the individual well of Novex® NuPAGE SDS-PAGE gel and

electrophoresis was carried out at 200 V and the resolved bands were visualized by staining with

Coomassie blue.

1.4.5 Elemental analysis

The elemental (C, N and S) spectra of AA and GEM conjugated BSA and native BSA was

analyzed via Flash 2000 Organic elemental analyzer (Thermo Scientific). Briefly, approximately

5 mg of sample was taken in a tin boat and analyzed using helium as the carrier gas and

elemental spectra was recorded and expressed as atomic percentage.

1.4.6 Circular dichroism (CD) assay

The conformational integrity of AA and GEM modified BSA and native BSA was determined

via far UV-CD spectroscopy (J-815; Jasco, Tokyo, Japan). Briefly, different samples (BSA, AA-

BSA, GEM-BSA and AA-GEM BSA conjugate) were placed in rectangular quartz cuvette of

path length 0.1 cm and scanned in the far UV region of 260–190 nm. The baseline correction

was done with distilled water and for each sample an average of three accumulations was taken

to obtain the CD spectrum.

1.4.7 Raman spectroscopy

Raman spectra of AA BSA, GEM BSA, AA-GEM BSA conjugate and native BSA were

obtained using Raman spectroscopy (ThermoScientific, Madison, USA) with a DXR 532 nm

laser. The conformation changes in the α-helix and ß-sheets of the lyophilized samples were

recorded over a range of 500-1700 cm-1 at room temperature (25 °C).

1.4.8 Differential scanning calorimetry (DSC) and Thermogravimetric analysis (TGA)

Thermal characteristics of BSA, AA, GEM, AA-BSA, GEM-BSA and AA-GEM BSA conjugate

were analyzed using DSC Mettler Toledo 821e TGA/SDTA (Mettler Toledo, Switzerland).

Empty aluminum pan was used to calibrate the instrument for baseline correction and each

sample of approximately 3–5 mg was kept in pan and heated in the range of 20−300 °C at a

scanning rate of 10 °C/min under inert nitrogen atmosphere. Further, the degradation profile of

AA-GEM BSA conjugate with temperature was also analyzed and compared with the native

BSA by using thermogravimetric analysis at the same temperature range.

1.4.9 Contact angle

Contact angle of AA-BSA, GEM-BSA, AA-GEM-BSA conjugates and native BSA were

measured by sessile drop method utilizing Drop Shape Analyser instrument (FTA 1000, First

Ten Angstroms, Virginia, USA). Different samples were mounted on double sided adhesive tape

adhered to a glass slide and a drop of liquid medium (Milli-Q water) was dispensed on their

surface and were captured by the FTA image analyser. The surface tension of Milli-Q water was

measured to be 73.1 ± 0.5 mN/m at 25 ± 2 °C. All measurements were performed in a controlled

environment conditions of 25 ± 2 °C/55 ± 5% RH. Contact angle was then calculated by fitting

mathematical expression to the shape of the drop and calculating the slope of the tangent to the

drop at the liquid-solid-vapor interface line.

1.4.10 Determination of critical aggregation concentration (CMC)

The CAC of the AA-GEM-BSA conjugate was determined by using pyrene as an extrinsic

probe.(3) Briefly, conjugate was dissolved in water to prepare stock solution and subsequent

dilutions were prepared through the stock solution from range of 0.5-100 µg/ml. Another stock

solution of pyrene was prepared in acetone and from this stock, 4 µg equivalent of pyrene was

added to all the conjugate dilutions. These dilutions were then scanned using Cary Eclipse

fluorescence spectrometer with the excitation range of 190 nm to 400 nm. The spectra bands, of

pyrene fluorescence, were determined at 334 nm and 339 nm respectively. The excitation

intensity ratio of I334/I339 was used to determine the CAC by plotting graph of intensity ratio vs

log concentration.

1.5 Experimental design

Response surface methodology is a group of statistical and mathematical method which is useful

for the modelling and analysing formulation problems. The main objective of this technique is to

optimize the response surface that is influenced by various process parameters. Response surface

methodology also quantifies the relationship between the controllable input parameters and the

obtained response surfaces. A Box-Behnken design (BBD) is a type of response surface design

that does not contain an embedded factorial or fractional factorial design and it has treatment

combinations that are at the midpoints of the edges of the experimental space and require at least

three continuous factors. These designs allow efficient estimation of the first- and second-order

coefficients because BBD often have fewer design points; they can be less expensive to do than

central composite designs with the same number of factors. However, because they do not have

an embedded factorial design, they are not suited for sequential experiments. The experimental

design and statistical analysis were performed using the Design Expert software 9.0.7.1 software.

A three factors three levels Box–Behnken experimental design. was used for optimization and

evaluate the relationship between the independent variables like pH (A), Homogenization

pressure (B), Drug loading (C), and dependent (responses) variables, i.e., particle size (Y1),

polydispersity index (Y2), and entrapment efficiency (Y3). As per the experimental runs, total 17

formulations had been prepared.

1.6 Optimization

In order to ascertain the optimum formulation, it is necessary to evaluate the effect of

formulation parameters and their interactions on the properties of the final product. The results of

the experimental design were analysed using Design-Expert software, which provided

considerable useful information and reaffirmed the utility of statistical design for conduct of

experiments. The selected independent variables including the pH, homogenization pressure, and

drug loading significantly influenced the observed responses for EE (%), particle size, and

polydispersity index which are presented in Table S 6. Polynomial equations involving the main

effect and interaction factors were determined based on estimation of statistical parameters such

as multiple correlation coefficient, adjusted multiple correlation coefficient, and the predicted

residual sum of squares generated by Design-Expert software. The statistical validation of the

polynomial equations was established by ANOVA provision available in the software.

Therefore, the optimum values of the variables were determined according to the obtained

experimental data using the Design-Expert software, based on the constrained criterion of

desirability presented in Table S 16. Response surface analyses plotted in three-dimensional

model graphs for depicting the effects of the predetermined factors on the response of the

entrapment- efficiency, particle size and PDI are shown in Figure S 6, based on the model

polynomial functions, to assess change in the response surface. The response surface plots were

used to study the interaction effects of 2 independent variables on the responses or dependent

variables, when a third factor is kept at constant level. When these plots were carefully observed,

the qualitative effect of each variable on each response parameter could be visualized.

Table S 5: Variables and their levels in the Box-Behnken design

Variables Lower level Upper level

pH 6 8

Homogenization pressure

(psi)

15000 20000

Drug loading (%) 5 15

Table S 6: Box-Behnken experimental design

Ru

n

Factors Responses

A;

pH

B;

Homogenizatio

n

Pressure (psi)

C;

Drug loading

(%)

Particle

Size

(nm)

PDI Entrapment

Efficiency

(%)

1 8 17500 5 162.35 0.328 55.21

2 7 15000 15 203.18 0.473 60.2

3 7 20000 5 176.31 0.328 44.87

4 8 15000 10 119.4 0.328 62.03

5 6 17500 15 318.2 0.473 48.22

6 7 15000 5 193.25 0.465 58.61

7 6 20000 10 302.37 0.338 37.82

8 7 17500 10 251.87 0.394 60.83

9 6 17500 5 311.98 0.528 38.37

10 7 17500 10 283.29 0.407 59.15

11 7 17500 10 239.13 0.438 60.58

12 7 17500 10 287.54 0.437 60.7

13 7 20000 15 182.39 0.282 65.26

14 7 17500 10 263.81 0.321 64.66

15 6 15000 10 328.44 0.528 50.17

16 8 20000 10 127.28 0.133 78.92

17 8 17500 15 172.96 0.165 76.3

1.7 Characterization of DTX loaded AA-GEM-BSA NPs

1.7.1 Freeze drying of DTX loaded AA-GEM-BSA NPs

To further enhance the storage stability, the developed AA-GEM-BSA NPs were lyophilized

using (Vir Tis, Wizard 2.0, New York, USA freeze-dryer) our previously patented stepwise

freeze-drying cycle with slight modification (4, 5). Different cryoprotectants viz., trehalose,

mannitol, and sucrose were screened at 5% w/v during preliminary screening and mannitol was

finalized based on the redispersibility index, and reconstitution score of the freeze-dried NPs.

Mannitol was further optimized for the efficient concentration between the range of 2.5−10%

w/v.

1.7.2 Powder X-ray diffraction analysis (PXRD)

The PXRD patterns of pure AA, GEM, DTX, BSA, Mannitol (MT), AA-GEM-BSA conjugate,

physical mixture (equal mixture of AA, DTX, GEM, BSA and MT) and DTX loaded AA-GEM-

BSA NPs were recorded on X-ray diffractometer (D8 Advanced Diffractometer, Bruker AXS

GmbH, Germany). The samples were scanned from 4° to 40° (2θ at a scan rate of 0.1° (2θ) /min)

and the obtained diffractograms were further studied with DIFFRAC plus EVA (ver.9.0)

diffraction software.

1.7.3 In vitro release

In vitro release profile of DTX from DTX loaded AA-GEM-BSA NPs was determined in

phosphate buffer saline pH 7.4 and pH 5.5, to mimic the pH of blood and tumor

microenvironment, respectively. Briefly, freeze dried NPs (equivalent to 500 μg of DTX) were

dispersed in 500 μL of release medium and filled into the dialysis bag and suspended in 20 ml of

phosphate buffer (pH 7.4 and 5.5) containing 0.1% Tween 80. Thereafter the vials containing

dialysis bag were kept in a shaker bath at 37°C and 100 rpm and at predetermined time points

(0.5, 1, 2, 4, 6, 8, 12, 24 and 48), 1 ml sample was withdrawn from the outer compartment and

replaced with an equal quantity of fresh buffer. The samples were analyzed by HPLC, and

cumulative % drug release was calculated.

1.7.4 In vitro hydrolysis in simulated fluids (GEM release)

In vitro hydrolysis of GEM from AA-GEM-BSA NPs was evaluated in phosphate buffered

saline (PBS) at pH 5.5 and pH 7.4, in the presence and absence of protease in order to simulate

the tumor microenvironment and systemic circulation, respectively. Briefly, NPs equivalent to 1

mg GEM, dispersed in 1 ml of the distilled water (containing 20 μl of 5 U/ml concentration of

enzyme) were taken in a dialysis bag (molecular weight cutoff 1000 Da) and placed in 5 ml of

media. At scheduled time intervals, samples aliquots (1 ml) were withdrawn and replaced with

the equal amount of fresh media to maintain the sink conditions. The free GEM released from

NPs was analyzed by validated HPLC method.

1.7.5 Plasma stability studies

In vitro degradation of GEM from NPs (equivalent to 0.5 mg GEM) into the metabolite (2′,2′-

difluorodeoxyuridine (dFdU)) was measured by incubating free GEM and NPs in plasma (1 ml)

for 24 h at 37°C. At predetermined time intervals, samples were withdrawn and analyzed by

validated bioanalytical method using HPLC.

1.7.6 Accelerated stability studies

Freeze dried NPs, with mannitol as a cryoprotectant, were assessed for accelerated stability

studies over a period of 6 months as per the protocol described in our earlier reports (6, 7). The

optimized NPs were kept in stability chamber with temperature of 25±2 °C and RH 60%±5%.

After 6 months, the nanoformulations were then evaluated for change in particle size.

1.8 Cells

1.8.1 Cytotoxicity

Cell cytotoxicity of free drugs and DTX loaded AA-GEM-BSA NPs in MCF-7 and MDA-MB-

231 cell lines was assessed via standard MTT as per our previously reported protocol (8).

Briefly, MCF-7 and MDA-MB-231 (10,000 cells/well) were seeded to 96-well tissue culture

plates (Costars, Corning Inc., NY, USA) and kept overnight. Following attachment, the cells

were incubated with fresh medium containing free drugs and NPs at a concentration of 0.1, 1, 5,

and 10 μg/ml (equivalent to free DTX) and further incubated for 48 and 72 h. After the

incubation period, the medium containing different treatments was aspirated and the formazan

crystals then solubilized with DMSO and the optical density (OD) was measured at 550 nm

using an ELISA plate reader (BioTek, USA). The cell viability was calculated by equation 2.

Relativecell viability= Absorbance(Sample)Absorbance (Control) (2)

1.8.2 DNA damage assay

The DNA damage potential of the free drugs and AA-GEM-BSA NPs was assessed as a function

of alterations in the levels of DNA damage marker (8-hydroxyguanosine (8-OHdG)) (9). Briefly,

cultured MCF-7 and MDA-MB-231 cells were exposed to varying concentrations of free drug

and NPs (0.1, 1, 5 and 10 µg/ml equivalent to DTX). After incubation for 12 h, cells were then

washed with HBSS and pelletized. Thereafter, the levels of 8-OHdG were evaluated using

ELISA kit (OxiSelect Oxidative DNA Damage ELISA Kit, STA-320) following the

manufacturer's instructions by taking DMSO treated cells as a negative control.

1.8.3 Nucleoside transporter (hNTs) and OATP1B3 inhibition

Dipyridamole was used as hNTs and OATP1B3 membrane transporter inhibitor to evaluate

transporters dependent membrane permeation of free drugs and NPs.(10-13) Briefly, MCF-7 and

MDA-MB-231 (10,000 cells/well) were seeded in 96-well plates and incubated with inhibitors

viz. dipyridamole (DIP) (10 mM, hNTs and OATP1B3 transporter inhibitor) for 1 h. Following

the incubation, the media was aspirated and the cells were incubated with GEM, DTX, their

combination (1:1 molar ratio) and DTX loaded AA-GEM-BSA NPs at concentrations of 0.1, 1,

10, and 20 μg/ml (equivalent to free DTX) for 24 h and IC50 was calculated by CalcuSyn 2.1

software.

1.8.4 Internalization pathways

Internalization pathways of AA-GEM-BSA NPs were assessed using clathrin and caveolae

endocytic inhibitors. Briefly, MCF-7 and MDA-MB-231 cells were pretreated with

chlorpromazine (CPZ) (clathrin-mediated endocytosis inhibitor; 10 mg/ml), genistein (GNT)

(caveolae mediated endocytosis inhibitor; 1 mg/ml), and combination of DIP, CPZ and GNT for

1 h at 37 °C.(14) After incubation, the media was aspirated and the cells were further incubated

with DTX loaded AA-GEM-BSA NPs (equivalent to 10 μg/ml of free C-6) for 2 h. The cells

were then washed with HBSS for three times to remove the extracellular particles and observed

under CLSM.

2 RESULTS

2.1 Result

2.1.1 Analytical method development and validation for Gemcitabine

2.1.1.1 Linearity and range

Standard calibration curve was constructed in the concentration range of 1-50 μg/ml, showing

good linearity with correlation coefficient of 0.999 and other validation parameters (Table S7).

Table S7: HPLC method validation parameters for in-vitro method

Parameter ValueRange 1-50 µg/mlLinearity (R2) 0.999±0.002Slope 72816.94±885.72Intercept 1907.92±60.30LOD 0.0027 µg/mlLOQ 0.0083 µg/mlValues are expressed as mean ± SD (n=6)

2.1.2 Bioanalytical method development for GEM

2.1.2.1 Linearity and range

The chromatogram of drug and internal standard (IS) was observed in single simultaneous run

when subjected to in-vivo method (Bio-analytical). The retention time was found to be 8.167 min

and 12.967 min for GEM and IS, respectively. Standard calibration curve was constructed in the

concentration range of 10-1000 ng/ml, showing good linearity with correlation coefficient of

0.9992 and other validation parameters (Table S8).

Table S8: HPLC method validation parameters for in-vivo samples

Parameter ValueRange 10-1000 ng/mlLinearity (R2) 0.9992±0.0321Slope 0.0241±0.0562Intercept 0.0546±0.0087Values are expressed as mean ± SD (n=6)

2.1.3 Analytical method development and validation for Docetaxel

2.1.3.1 Linearity and range

Standard calibration curve was constructed of seven concentrations in the range of 10-1000

ng/mL, which shows good linearity with correlation coefficient of 0.9981 and other validation

parameters shown in Table S9.

Table S9: In-vitro HPLC method validation parametersParameter Value of DTXLinearity range 10-1000 ng/mLLinearity (R2) 0.9981±0.00753Slope 23654.5±854.13Intercept 3323.5±3673.969LOD 0.2508 µg/mLLOQ 0.7603 µg/mL*Values are expressed as mean ± SD (n=6)

2.1.4 Bioanalytical method development for DTX

2.1.4.1 Linearity and range

The chromatogram of drug and internal standard (PTX) was observed in single simultaneous run

when subjected to in-vivo method (Bio-analytical). Standard calibration curve was constructed in

the concentration range of 200-10000 ng/ml, showing good linearity with correlation coefficient

of 0.998 and other validation parameters (Table S8).

Table S10: HPLC in-vivo method validation parameters

Parameter SpecificationRange 200ng-10μgLinearity (R2) 0.998±0.0005Slope 0.803±0.063Intercept 0.073±0.0298Values are expressed as mean ± SD (n=6)

2.2 Characterization of DTX loaded AA-GEM-BSA conjugate

2.2.1 Degree of modification (TNBS and titration method)

The quantitative estimation of amino groups in the L-lysine in the concentration range of 1-20

μg/mL was evaluated and used as a reference. The molar concentration of free amino groups in

BSA and AA-GEM-BSA conjugate was found to be 182.7 ± 9.1 μmol and 158.3 ± 9.7 μmol,

respectively, while, the free carboxylic group in BSA and AA-GEM-BSA conjugate was found

to be 295.9 ± 13.7 μmol and 271.3 ± 9.7 μmol, respectively. The corresponding reduction of the

amino and carboxylic groups in AA-GEM-BSA conjugate as compared to BSA was a result of

conjugation of both AA and GEM with BSA via amide bond. Assuming amino and carboxylic

groups of BSA as 100%, the degree of conjugation of AA and GEM was found to be 13.35 and

8.31 %, respectively. (Table S 11)

Table S 11: Degree of modification determined via 2,4,6-trinitrobenzene sulfonic acid (TNBS)

method

BSA (μmol) GEM-BSA (μmol)

Amine group determination

Free amino group concentration 182.7 ± 9.1 158.3 ± 9.7

Degree of modification (DM) % 13.35%

Carboxylic group determination

Free carboxylic group concentration 295.9 ± 13.7 271.3 ± 9.7

Degree of modification (DM) % 8.31%

Values are presented as mean ± SD (n=6).

2.2.2 Fluorescence spectroscopy

AA-GEM-BSA conjugate resulted in about 67.25% quenching of fluorescence intensity (counts)

of 15215 ± 732 as compared to 46471 ± 783 of native BSA. The fluorescence quenching

observed in case of AA-GEM-BSA conjugate may be assigned to some conformational changes

in BSA following the covalent conjugation.

Table S 12: Intrinsic tryptophan fluorescence emission counts of BSA and drug-BSA conjugates

(λex = 280 nm)

S. No. Samples Fluorescence intensity (Counts)

1. BSA 46471 ± 783

2. GEM-BSA conjugate 19734 ± 642

3. AA-BSA conjugate 16542 ± 659

4. AA-GEM-BSA conjugate 15215 ± 732

Values are presented as mean ± SD (n=6).

2.2.3 MALDI-TOF

AA-GEM-BSA conjugate exhibited significant increase in molecular weight as compared to AA-

BSA, GEM-BSA conjugates and native BSA (Figure S 1 A). The center of mass distribution of

native conjugate was found to be m/z 66206.31, while, after conjugation with both AA and GEM

the center of mass curve was shifted to m/z 71218.66.

2.2.4 SDS gel electrophoresis

In line with the MALDI-TOF results, SDS-PAGE demonstrated remarkable increase in

molecular weight of AA-GEM BSA conjugate as compared to AA-BSA, GEM-BSA and native

BSA (Figure S 3 B).

2.2.5 Elemental analysis

The compositions of AA-GEM-BSA, AA-BSA, GEM-BSA conjugates and BSA were evaluated

via elemental analysis. Elemental analysis of BSA was found to be 13.61%, 45.20%, 7.18% and

1.45% in case of N, C, H and S, respectively. As evident from Table S 13, the percentage

increase in N (in case of GEM-BSA and AA-GEM-BSA conjugates) and C (in case of GEM-

BSA, AA-BSA and AA-GEM-BSA conjugates) content demonstrated the synthesis of conjugate.

Table S 13: Percentage composition of different elements

S. No. Name of Sample Nitrogen % Carbon % Hydrogen % Sulfur %

1 BSA 13.61 45.20 7.18 1.45

2 GEM-BSA 13.96 47.9 6.95 1.14

3 AA-BSA 13.54 48.27 7.04 1.11

4 AA-GEM-BSA 13.88 51.78 7.11 0.98

2.2.6 Circular dichroism (CD) assay

CD spectra of BSA and conjugates are shown Figure S 3 C. AA-GEM-BSA conjugate exhibited

α helix, ß sheets and turns percentage of 26.4, 9.4 and 29.9%, while the native BSA

demonstrated 26.2, 31.6 and 18.1, respectively (Table S 14).

Table S 14: Percentage of structural components of BSA and GEM-BSA

S. No. Sample Helix% Beta% Turn%

1. BSA 26.2 31.6 18.1

2. AA-BSA conjugate 17.7 47.4 10.45

3. GEM-BSA conjugate 22.8 19.2 26.8

4. AA-GEM-BSA 21.4 9.4 29.9

conjugate

2.2.7 Raman spectroscopy

Raman spectra of native BSA and BSA conjugates were measured using Raman microscope

(ThermoScientific, Madison, USA) with a DXR 532 nm laser. Figure S 3 D and Table S 15

demonstrates decrease in intensity ratio (from 0.678 to 0.366) of I934/I1003 and an increase in

intensity ratio (from 0.697 to 1.158) of I1246/I1337, which depicts the conformational

transformation in α-helix and β-sheets after conjugation of GEM and AA with the BSA.

Table S 15: Intensity ratio (I934/I1003 and I1246/I1337) of BSA and GEM-BSA in solid state Raman

spectroscopy

S. No. Samples I934/I1003 I1246/I1337

1. BSA 0.678 0.697

2. GEM-BSA conjugate 0.541 0.859

3. AA-BSA conjugate 0.440 1.128

4. AA-GEM-BSA conjugate 0.366 1.158

Figure S 3: (A) MALDI-TOF spectra of BSA, AA-BSA, GEM-BSA and AA-GEM-BSA

conjugate; (B) SDS PAGE analysis of BSA (lane1), GEM-BSA (lane 2), AA- BSA (lane 3) and

AA-GEM-BSA (lane 4); (C) Overlay CD spectra of BSA and BSA conjugate and (D) Raman

spectra of native BSA and different BSA conjugates

2.2.8 Differential scanning calorimetry (DSC) and Thermogravimetric analysis (TGA)

DSC thermograms of AA, GEM, BSA, physical mixture, AA-GEM-BSA conjugate are

presented in Figure S 4 A. GEM and physical mixture exhibited a sharp endotherm peak at

~273ºC corresponding to the melting point of GEM and a broad peak of AA was obtained at

~30ºC owing to its amorphous properties. While, BSA, AA-GEM-BSA exhibited two broad

peaks at ~60ºC and ~220ºC. Furthermore, TGA analysis demonstrated biphasic degradation

profile of BSA conjugates and native BSA. As depicted in Figure S 4 B, initial weight loss in the

range of ~4-10% was observed from room temperature to ~100 ºC. After, initial weight loss

phase, a plateau or stable phase was observed from ~110 ºC to ~220 ºC followed by second

degradation phase to 75.51% weight loss in case of native BSA, while, BSA conjugates

demonstrated a shorter stable phase followed by significant higher degradation up to 64.14%,

67.41% and 60.80% GEM-BSA, AA-BSA, AA-GEM-BSA conjugates in comparison with BSA,

respectively.

2.2.9 Contact angle analysis

Qualitative and quantitative estimation of wetting behavior of the developed conjugates was

assessed via contact angle analysis. As evident from the Figure S 4 C and D, the water droplet

demonstrated higher affinity towards GEM-BSA conjugate, and in opposite, reduced affinity

with AA-BSA conjugate as compared to native BSA. While, the water droplet affinity was

regained when both AA and GEM were conjugated with BSA with contact angle of 109.75

± 2.63°.

Figure S 4: (A) DSC and (B) TGA thermograms of BSA and BSA conjugate; (C) Qualitative

and (D) Quantitative contact angle evaluation/Water droplet profile on films of (a) BSA, (b) AA-

BSA conjugate, (c) GEM-BSA conjugate, (d) AA-GEM-BSA conjugate

2.2.10 Critical aggregation concentration (CAC)

CAC was evaluated of the synthesized AA-GEM-BSA conjugate, by using fluorescence

spectroscopy taking pyrene as a probe. As evident from Figure S 5, CAC was found to be 5.636

µg/mL.

Figure S 5: CAC determination of AA-GEM-BSA conjugate from pyrene excitation

spectra

2.2.11 Effect on PDI

PDI for various factor levels combinations shown in Table S 16. In Figure S 6, the effect of

varying the amount of different factors on the PDI (Y2) was studied when the other factor was

kept constant. The effect can be explained by the following quadratic equation:

PDI = +0.37 -0.11 * A -0.089 * B -0.032 * C eq. (2)

The positive value before a factor in the regression equation indicates that the response increases

with the factor and vice versa. The value of the correlation coefficient (R2) of above equation

was found to be 0.8559, indicating a good fit.

2.3 Experimental design

2.3.1 Effect on Particle Size

The particle size for various factors level combinations are shown in Table S 16. The most

significant factor contributing to the variation in pH was A as evident from the value of the

coefficient. The factor A showed that a negative effect on the particle size which means an

increase in the value of A will cause a decrease in the value of particle size. The effect can be

explained by the following quadratic equation:

Size = +265.13-84.88* A-6.99* B+4.10* C+8.49* AB+1.10* AC-0.96* BC+3.42* A2-49.17*

B2-27.17* C2 eq. (1)

The positive value before a factor in the regression equation indicates that the response increases

with the factor and vice versa. The value of the correlation coefficient (r2) of above equation was

to be 0.9637, indicating a good fit. In Figure S 6. the effect of different variables on the particle

size (Y1) was studied when the other factor was kept constant.

2.3.2 Effect on Entrapment Efficiency

Entrapment efficiency for various factors level combinations shown in Table S 16. The effect

can be explained by the following quadratic equation:

%EE = +61.18 +12.23 * A -0.52 * B +6.62 * C +7.31 * AB +2.81 * AC +4.70 * BC -3.33 * A2 -

0.62 * B2 -3.33 * C2 eq. (3)

The value of the correlation coefficient (R2) of above equation was found to be 0.9721,

indicating a good fit.

Table S 16: Model summary

Variables R-

Squared

Adj R-

Squared

Pred R-

Squared

Adeq

precision

Particle Size (nm) 0.9637 0.9169 0.7426 12.959

PDI 0.8559 0.8227 0.7559 17.559

% Entrapment efficiency

(%EE)

0.9721 0.9363 0.6741 17.876

Figure S 6, the effect of varying the different factors on the entrapment efficiency (Y3) was

studied when other factor was kept constant. The pH (A) had a significant and negative effect on

%EE as revealed by the positive value in the quadratic equation. The result showed that the

entrapment efficiency increased as the pH increases.

2.3.3 Optimization and Validation

The desirability function (desirability=0.945) was probed using Design-Expert software to

acquire the optimized formulation. Figure S 6 (d) shows the overlay plot. The optimum

formulation was based on the set criteria of maximum entrapment efficiency, minimize particle

size and PDI. Therefore, a new batch of formulation with the predicted levels of formulation

factors was prepared to confirm the validity of the optimization procedure. The composition of

optimized formulation was achieved with the pH 8, drug loading 9% and homogenization

pressure of 20000 psi which fulfils the requirements of optimization. From the experimentation,

162.5±8.2 nm particle size, 0.127±0.09 PDI and 75.50±3.26 % entrapment efficiency were

observed which were in good agreement with the predicted values (134.06±19.75 nm particle

size, 0.148±0.048 PDI and 73.30±2.85% entrapment efficiency) and within 95% confidence

interval.

(a) (b)

(c) (d)

Figure S 6: (a-c) Response surface plot showing effect of drug loading and pH on (a) particle

size, (b) PDI, (c) entrapment efficiency; (d) Overlay plot

2.4 Characterization of DTX loaded AA-GEM-BSA NPs

2.4.1 Freeze drying of DTX loaded AA-GEM-BSA NPs

Mannitol and trehalose (5% w/v) resulted in the formation of intact, voluminous, fluffy and easy

to redisperse cake, while the cake was not intact in case of sucrose cryoprotectants. Different

properties viz. physical appearance, reconstitution nature and size ratio (before and after freeze

drying) of freeze dried NPs are shown in Table S 17 and Table S 18.

A significant increase in particle size was observed following the freeze drying without any

cryoprotectants while the difference was insignificant in case of different concentrations of

different cryoprotectants. Based on the appearance, redispersibility index and reconstitution

score mannitol (5% w/v) was selected as optimized one.

Table S 17: Freeze drying of DTX loaded AA-GEM-BSA NPs using different cryoprotectants at

5% concentration

Concentration (%)

Initial 5 %

Mannitol

Size (nm) 164.8±8.03 173.0±8.2

Ri - 1.05±0.04

RS - ***

Sucrose

Size (nm) 164.8±8.03 234.01±13.9

Ri - 1.42±0.09

RS - *

Trehalose

Size (nm) 164.8±8.03 186.2±8.6

Ri - 1.12±0.07

RS - ***

Table S 18: Freeze drying of DTX loaded AA-GEM-BSA NPs using mannitol at different

concentrations.

Concentration (%)

Initial 0 2.5 5 10

Mannitol

Size (nm) 164.8±8.03 ND 178.3±10.4 173.0±8.2 169.7±8.6

Ri - ND 1.09±0.06 1.05±0.04 1.03±0.05

RS - * ** *** ***

Ri-Redispersibility index, RS-Reconstitution score, ***redispersible within 20 sec with mere mixing, **

redispersible within 1 min, ND-not determined due to incomplete redispersion of cake. *reconstitution requires

high shear vortexing for 2min, but the cake was not completely redispersed, a- Dense white partially

cracked cake, b- Dense white partially cracked cake, c- Dense white intact cake. Values are presented as mean ± SD

(n=3).

2.4.2 Powder X-ray diffraction analysis (PXRD)

Figure S 7 demonstrated characteristic diffraction peaks of DTX and GEM in case of free drugs

and physical mixture of mannitol, DTX, GEM, conjugate. However, characteristic peaks of

GEM were disappeared in DTX loaded AA-GEM-BSA NPs, which confirmed the absence of

crystallinity of drug owing to GEM conjugation and DTX encapsulation within the NPs.

Figure S 7: PXRD diffractogram of GEM, DTX, AA, mannitol, AA-GEM-BSA conjugate,

physical mixture of BSA, GEM, AA, mannitol and DTX loaded AA-GEM-BSA NPs

2.4.3 In vitro release studies

Figure S 8 exhibits release profile of DTX at pH 7.4 and 5.5. The amount of DTX released at pH

5.5 was slightly higher as compared to pH 7.4. The DTX released from AA-GEM-BSA NPs

showed biphasic release pattern with an initial burst release of 23.25 and 26.23% at pH 7.4 and

5.5, respectively, in 4 h followed by sustained release up to 48 h. The cumulative drug release

was fitted into different release models namely zero order, first order, Higuchi’s square root plot

and Hixson Crowell cube root plot. Among different release models, Higuchi’s square root

model demonstrates correlation coefficient (r2) values close to unity (0.994) and thus selected as

an order of release.

Figure S 8: In vitro release profile of DTX from AA-GEM-BSA NPs at pH (A) 5.5 and (B) 7.4

2.4.4 In vitro hydrolysis of GEM in simulated media

As evident from the Figure S 9 (I), rate of hydrolysis was little higher at pH 5.5 in comparison

with pH 7.4. Similarly, the rate of hydrolysis was higher in presence of proteases at both the

tested pH conditions.

2.4.5 Plasma stability studies

The plasma stability of GEM in case of NPs were evaluated by estimating the percentage of free

GEM and its metabolite (dFdU). As evident from the Figure S 9 (II) NPs demonstrated enhanced

stability and controlled release of GEM in plasma. NPs demonstrated approximately 24% of

initial GEM content after 24 h incubation in plasma. While, GEM degradation to dFdU was

found to be higher in case of free GEM with approximately 64% of dFdU level in 24 h.

Figure S 9: (I) In vitro release of GEM from NPs at pH (A) 7.4 and (B) 5.5 in the absence and

presence of proteases; (II) Percent of dFdU and GEM (*) following incubation of (a) free GEM,

(b) AA-GEM-BSA NPs in the presence of plasma. Values are expressed as Mean ± SD (n = 3)

2.4.6 Accelerated stability studies

Accelerated stability studies were done for 6 months at 25 ± 2 °C and RH 60% ± 5% as per the

ICH guidelines. Freeze dried AA-GEM-BSA NPs showed no change in terms of shrinkage of

cake or any other change in physical appearance. Insignificant (p > 0.05) increase in the particle

size (from 159.7±8.94 to 168.34±10.62 nm) and PDI (from 0.136 ± 0.027 to 0.177 ± 0.032)

(Table S 19) were noted after the 6 months of testing period.

Table S 19: Characterization of formulation after 6 months of accelerated stability studies

Parameters Initial Final

Particle size (nm) 159.7±8.94 168.34±10.62

PDI 0.136 ± 0.027 0.177 ± 0.032

Ease of reconstitution *** ***

Physical appearance Intact fluffy cake Intact fluffy cake

*** redispersible within 20 sec with mere mixing. Values are presented as mean ± SD (n=6)

2.5 Cells

2.5.1 Cytotoxicity studies

In vitro cell cytotoxicity of free drugs (DTX, GEM), their combination (DTX+GEM), blank AA-

GEM-BSA NPs and DTX loaded AA-GEM-BSA NPs were evaluated on MCF-7 and MDA-MB-

231 cell lines, which revealed significantly enhanced cytotoxicity of DTX loaded AA-GEM-

BSA NPs as compared to free drugs, in both time and concentration dependent manner (Figure S

10 (I) and (II)).

2.5.2 DNA damage assay

The DNA damage assay further revealed that observed cytotoxicity of the free drugs and NPs is

facilitated by DNA damage. Significantly, higher levels (p < 0.001) of 8-OHdG were noted in

case DTX loaded AA-GEM-BSA NPs as compared to that of free drugs and their Figure S 10

(III).

Figure S 10: (I) Concentration and time-dependent cytotoxicity profile of free drugs and NPs in

(I) MCF-7 and (II) MDA-MB-231 cell lines after (A) 24 (B) 48 and (C) 72 h treatment and (III)

8-OHdG levels following the different treatments in (A) MCF-7 and (B) MDA-MB-231 cell

lines

2.5.3 Nucleoside transporter (hNTs) and OATP1B3 inhibition

Nucleoside transporter inhibition study was performed to investigate the dependence of GEM

and DTX on the hNTs and OATP1B3, respectively, for their cellular uptake and therapeutic

effect. MCF-7 and MDA-MB-231 cell lines were incubated with dipyridamole (hNTs and

OATP1B3 inhibitor), before the treatment with DTX, GEM and AA-GEM-BSA NPs. Thereafter,

the IC50 values were obtained in the absence and presence of dipyridamole. The presence of

dipyridamole increased the IC50 value by 5.92, 10.42 and 5.26-folds in MCF-7 and 6.90, 9.51 and

6.13-folds in MDA-MB-231 for DTX, GEM and combination of DTX and GEM. However, this

difference was found to be insignificant in case of AA-GEM-BSA NPs (p>0.05). Thus, DTX

loaded AA-GEM-BSA NPs aids in overcoming the drug resistance in both MCF-7 and MDA-

MB-231 cell line, when treated with dipyridamole (Table S20).

2.5.4 Internalization pathways

The internalization pathways of the developed NPs were also investigated by measuring the

relative resistance towards the drug treatment (Table S20). Insignificant increase in IC50 values,

in case of free drug treatment, was found when MCF-7 and MDA-MB-231 cells were incubated

with GNT and CPZ. In contrast, significantly higher increase in IC50 values, in case of DTX

loaded AA-GEM-BSA NPs treatment was found when the cells were pre-incubated with CPZ

(relative resistance of 4.14 and 5.07 for MCF-7 and MDA-MB-231, respectively). Interestingly,

the on pre incubation of combination of CPZ, GNT and DIP, the relative resistance in case of

free drugs were found be slightly higher as compared to DIP pre-incubation alone. Whereas, in

case of NPs treatment, the relative resistance significantly increased (approx. 2-3 folds) as

compared to individual inhibitors, which further validates the dependence of NPs on both

clathrin and caveolae mediated cellular internalization pathways.

Table S20: Effect of inhibitor on cytotoxic activity of free drugs and NPs in MCF-7 and MDA-

MB-231 cell lines.

Samples MCF-7

IC50

(Control

IC50 (DIP) Relative

Resistanc

IC50

(GNT)

Relative

Resistanc

IC50

(CPZ)

Relative

Resistanc

IC50 (DIP+

CPZ

Relative

Resistanc

) e e e +GNT) e

DTX 4.46±0.4

2

26.43±1.4

7

5.92 4.83±0.36 1.08 4.73±0.26 1.06 31.68±1.87 7.10

GEM 9.35±0.3

5

97.49±2.7

5

10.42 9.98±0.63 1.06 10.03±1.2

7

1.08 107.96±3.16 11.54

DTX+GE

M

3.87±0.2

3

20.36±1.1

8

5.26 4.24±0.25 1.10 4.16±0.25 1.08 28.89±1.51 7.46

DTX

loaded

BSA NPs

3.53±0.2

5

3.48±0.19 0.98 11.47±0.7

6

3.24 16.65±0.8

1

4.71 29.32±1.86 8.03

AA-GEM-

BSA-NPs

6.35±0.2

1

6.89±1.38 1.08 20.72±1.0

3

3.12 26.73±1.1

8

4.21 52.81±3.74 8.31

DTX

loaded AA-

GEM-

BSA-NPs

2.57±0.1

6

2.74±0.21 1.06 7.85±0.52 3.05 10.64±0.6

2

4.14 23.74±1.56 9.23

Samples MDA-MB-231

IC50

(control)

IC50 (DIP) Relative

Resistanc

e

IC50

(GNT)

Relative

Resistanc

e

IC50

(CPZ)

Relative

Resistanc

e

IC50 (DIP+

CPZ+GNT)

Relative

Resistanc

e

DTX 4.25±0.2

6

29.35±1.3

1

6.90 4.52±0.25 1.06 4.62±0.36 1.09 34.92±1.69 8.21

GEM 9.94±0.6

3

94.62±3.3

1

9.51 11.88±1.1

6

1.19 10.14±1.2

6

1.02 106.75±3.45 10.73

DTX+GE

M

3.25±0.2

5

19.92±1.2

5

6.13 3.93±0.24 1.21 3.86±0.18 1.19 28.24±1.43 8.69

DTX

loaded

BSA NPs

3.41±0.2

2

3.50±0.19 1.02 12.01±0.7

6

3.52 18.94±1.3

9

5.55 32.52±1.58 9.53

AA-GEM-

BSA-NPs

6.43±0.3

5

6.78±0.31 1.05 22.36±1.8

9

3.48 27.15±2.1

4

4.22 52.93±2.28 8.23

DTX

loaded AA-

GEM-

BSA-NPs

2.12±0.1

3

2.35±0.16 1.10 8.25±0.61 3.89 10.75±0.5

5

5.07 25.28±0.63 11.92

Values are presented as mean ± SD (n = 3)

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