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Vitamin B6 Tethered Endosomal pH Responsive Lipid Nanoparticles for Triggered Intracellular Release of Doxorubicin Shweta Sharma, Ashwni Kumar Verma, Jyotsana Singh, B Venkatesh Teja, Naresh Mittapelly, Gitu Pandey, Sandeep Urandur, Ravi Shukla, Rituraj Konwar, and Prabhat Ranjan Mishra ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08958 • Publication Date (Web): 14 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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ACS Applied Materials & Interfaces

Vitamin B6 Tethered Endosomal pH Responsive Lipid Nanoparticles for Triggered Intracellular Release of Doxorubicin Shweta Sharma†#, Ashwni Verma†, Jyotsana Singh‡#, B Venkatesh Teja†, Naresh Mittapelly†#, *

Gitu Pandey†#, Sandeep Urandur†, Ravi Shukla†, Rituraj Konwar‡, Prabhat Ranjan Mishra†# †Division of Pharmaceutics, CSIR-Central Drug Research Institute, Lucknow, India ‡Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, India #Academy of scientific and innovative research, New Delhi, India

*Corresponding Author Dr. P.R. Mishra Ph.D Division of Pharmaceutics, Preclinical South PCS 002/011, CSIR-Central Drug Research Institute B.S. 10/1, Sector-10, Jankipuram Extension, Sitapur Road, Lucknow-226031, India Phone no +91-522-2612411(4537) Fax no +91-522-2623405 E-mail: [email protected] [email protected]

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Abstract: This study reports the development of Vitamin B6 (VitB6) modified pH sensitive charge reversal nanoparticles for efficient intracellular delivery of Doxorubicin (DOX). Herein, VitB6 was conjugated to stearic acid and the nanoparticles of the lipid were formulated by solvent injection method (DOX-B6-SA-NP). Due to the pKa (5.6) of VitB6, DOX-B6-SA-NP showed positive charge and enhanced release of DOX at pH 5. Confocal microscopy illustrated that DOX-B6SA-NP treatment keep higher DOX accumulation inside the cells that conventional pH insensitive lipid nanoparticles (DOX-SA-NP). The cationic charge of nanoparticles subsequently facilitated the endosomal escape and promoted the nuclear accumulation of DOX. Furthermore, in vitro cytotoxicity, apoptosis, cell cycle arrest and mitochondrial membrane depolarization studies supported the enhanced efficacy of DOX-B6-SA-NP in comparison to free DOX and DOX-SA-NP. Intravenous pharmacokinetics and bio-distribution investigations indicated that pH sensitive nanoparticles can significantly prolong the blood circulation time of DOX in biological system and increase the drug accumulation to tumor site. Consequent to this DOX-B6SA-NP also exhibited much enhanced therapeutic efficacy and lower toxicity in tumor-bearing rats compared to free DOX. The reduction in toxicity was confirmed by histological and survival analysis. In conclusion, these results suggest that the VitB6 modified charge reversal nanoparticles can be a novel platform for the successful delivery of anticancer drugs.

Keywords: Stearic acid; proton sponge effect ; LA-7 cells ; pyridoxine ; charge reversal ; Vitamin-B6 transporting membrane carrier (VTC);


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1. Introduction In the past decade nanoparticles mediated drug delivery have shown great promise in enhancing the outcome of chemotherapy and that is due to their improved pharmacokinetic and biodistribution profiles which leads to improved accumulation of drug at tumor site especially through enhance permeation retention (EPR) effect1. Although, this EPR effect enhance the accumulation of nanoparticles (NPs) at tumor site

2

but their poor cellular internalization and

insufficient intracellular drug release hampers the efficacy and therefore pose the biggest challenge for nanoparticle mediated delivery. The dilemma of extracellular stability versus intracellular drug release via the frequently used delivery system has always been a challenge3. To overcome the challenges, development of environment-responsive nanoparticles have been attempted which can simultaneously enhance the stability in blood and can improve the drug availability inside the tumor cells by releasing the payload in presence of stimuli. Several kinds of delivery systems which are responsive to different type of stimuli (e.g., pH, redox, enzymes, or others) have already been developed 4-6. Out of the several stimuli responsive delivery systems pH-responsive ones are the most frequently used, as pH varies significantly in biological system for example the tumor extracellular environment is much lower (pH 5.8–6.5) than that of the blood (pH 7.4), and the pH values of endosomes are even lower (pH 5.0–5.5)

7-8

. Moreover pH

sensitivity of the nanoparticles also plays an important role in endosomal lysis or endosomal escape for effective intracellular delivery 9. Till now several delivery systems responsive to intracellular pH have been developed. These systems often work by stimulating the release of drug at acidic pH of endosomes. However several reports suggest that only triggered release at endosomal pH is not enough for maximum efficacy of nanoparticles. This is because nanoparticles usually enter the cells through adsorptive endocytosis (non-specific) or receptor-mediated endocytosis (specific) and if not escaped from endo/lysosome they eventually get degraded or digested by the endosomal enzymes

10

. This

degradation often results in limited delivery of drugs to the intracellular targets. Therefore, development

of delivery system

which

can

facilitate drug escapement

from

the

endosomal/lysosomal environment is very important11 12. To achieve this in the present work we propose the development of pH responsive nanoparticle that not only trigger the release of drug at acidic pH but also facilitate the process of endosomal escape by generation of cationic charge. This cationic charge absorb the protons of acidic environment by means of specific chemical 3 ACS Paragon Plus Environment


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groups and then cause swelling/or rupture of the endo/lysosome

13

. Moreover these charge

reversal particle also propose a solution to the dilemma of surface charge of particles suitable for systemic administration and tumor targeting. In general particles with negative charge are considered suitable for systemic administration because they avoid uptake by reticuloendothelial system (RES) but at the same time this negative charge of particles hampers the binding or internalization to target cells. In contrast to this the positive charged particle are not suitable for systemic administration because of their rapid uptake by RES system but at the same time their positive charge support the direct uptake and endosomal escape by tumor cells. Therefore the charge reversal nanoparticles present best approach for systemic administration and tumor targeting because they remain negatively charge at blood pH 7.4 thereby reduce the uptake by RES system but become positive charge at tumor site thereby facilitates the uptake and endosomal escape

14 15

. Currently most of the reports which present pH responsive system

involve the modification of one or another polymer whereas in comparison to this lesser approaches have been used for the preparation of pH sensitive lipid nanoparticles. Therefore it is highly desirable to exploit simple and facile strategies for development of smart lipid based nano-carriers which can overcome limitation of polymeric systems as well as can surpass the multiple biological barriers successively to release the encapsulated drug intracellularly 16. OH

OH HO

OH

HO

pH 5.6 H3C

H3C

N

N H

Here in this investigation Vitamin B6 (VitB6, also called as pyridoxine HCL) having pKa 5.6 has been investigated as pH sensitive functional group. Due to its pKa, VitB6 is completely protonated and cationic below pH 5.6. This cationic charge then interacts with negativelycharged endosomal membranes, induces influx of water and ions and eventually brings about endosome destabilization and drug release. To the best of our knowledge, such interesting property of VitB6 has not been reported in the literature before. In addition to this VitB6 is also known to participate in enhancing the cellular uptake of delivery systems inside the cells by means of specific receptors. In general, tumor cells require high levels of vitamins and therefore 4 ACS Paragon Plus Environment

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their entry is facilitated inside the tumor cells by mean of specific receptors. This increase in uptake of VitB6 also mediates entry of VitB6-coupled molecules by Vitamin-B6 transporting membrane carrier (VTC) mediated entry into the tumor cells

17

. Previous also, VitB6 has been

shown to facilitate the cellular uptake of small peptides as well as nanoparticles through VTCs 1819

.

20

. Here in order to develop pH sensitive drug delivery systems (DDS) with excellent

biocompatibility and tumor targeting ability, lipid nanoparticles were developed based on conjugation of VitB6 to stearic acid. The conjugation of VitB6 (pH responsive moiety) on the terminal end of stearic acid, would allow the nanoparticles to maintain a negative charge at physiological pH, but quickly switch to a positive charge in weak acidic conditions. This chargereversible property could not only improve the stability of nanoparticles in blood circulation but also provide pH triggered drug release property and proton sponge effect which facilitate intracellular drug release. In addition to the pH sensitive property incorporation of VitB6 also participates in enhancing the accumulation of drug at tumor site by its targeting ability to VTC presence on tumor cells. LA 7 cells induced mammary adenocarcinoma model was used to evaluate the efficacy of nanoparticles. LA 7 cells are the cancer cells isolated from mammary adenocarcinoma induced in rats using 7,12-Dimethylbenz[a]anthracene (DMBA). These cells have the property of self renewal and undergo differentiation as stem cells. They are breast cancer cells having the property of generating tumor with single cell. These cells closely resembles metastatic breast cancer cells MDA-MB 231 derived from human patients and therefore could be used as a suitable in vivo model for evaluation of the anticancer efficacy of formulations 21-22. 2. Materials and Methods 2.1. Materials Stearic

acid,

Vitamin

B6

(pyridoxine

HCL),

4-(dimethylamino)pyridine

(DMAP),

dicyclohexylcarbodiimide (DCC, 99%) , N-hydroxysuccinimide (NHS, 99%) , 6-Diamidino-2phenylindole (DAPI), MTT (3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide), fluorescein isothiocyanate (FITC), Propidium iodide (PI), RNAse, were purchased from SigmaAldrich (St Louis, MO, USA). Doxorubicin hydrochloride (DOX.HCl) was gift sample form Fresinius kabi (Delhi, India). Lysotracker red was purchased from Fischer scientific, whereas Annexin-V apoptosis kit was from Merck. HPLC solvents such as Acetonitrile and Methanol were purchased from Merck (India). All other chemical reagents were of analytical grade and 5 ACS Paragon Plus Environment


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obtained from commercial sources. Triple distilled water (TDW) was prepared from Milli Q system (Millipore, Bedford, USA). All other chemicals were of analytical grade and used as such. 2.2. Methods Synthesis and characterization of Vitamin B6-Stearic acid (VitB6-SA) conjugate The conjugate between Stearic acid and Vit B6 (1:1 mole ratio) was prepared by esterification reaction performed in the presence of DCC and DMAP 23. In brief the stearic acid (1 Mole) was dissolved in DMF (2ml, anhydrous) and activated using 1.2 equivalents of DCC at 4°C for 2 h under the inert atmosphere of nitrogen. The activated stearic acid was then reacted with hydroxyl group of VitB6 (1 Mole) in the presence of 1.5 moles of DMAP under the inert conditions for another 24 hr at room temperature. The precipitate dicyclohexyl urea was removed by filtration and formed conjugate was precipitated by using Milli water. The reaction product stearic acid conjugated vitamin B6 (VitB6-SA) was washed several times with Milli Q and brine solution to remove the by product. The conjugate was lyophilized and characterized using 1H-NMR. Preparation of nanoparticles (DOX-B6-SA-NPs, DOX-SA-NPs) Before preparation of nanoparticles, DOX.HCl in water was converted to free DOX base by adding excess amount of triethylamine (TEA). TEA neutralized the HCl whereas free DOX was extracted in chloroform 24. After this, DOX loaded VitB6 conjugated stearic acid (DOX-B6-SANPs) were prepared by solvent injection method 25. For preparation of nanoparticles, VitB6-SA was dissolved in ethanol along with DOX and this ethanol solution is then injected into aqueous phase containing 0.2% w/v Tween 80. This suspension was then probe-sonicated for 5 min at 20% amplitude and the ethanol was evaporated under nitrogen stream. The formed dispersion of lipid nanoparticles was stirred under mechanical stirrer for 1 hr at 600 rpm to get uniform nanoparticulate dispersion. DOX loaded plain stearic acid NPs (DOX-SA-NPs) were also prepared by using similar protocol except that in place of VitB6-SA, plain stearic acid was used. Physicochemical characterization of different formulations and their encapsulation efficiencies The particle size, poly-dispersity index (PDI) and zeta potential were measured using Nano Zs 2000 (Nano ZS, Malvern instrument,UK) which works on dynamic light scattering principle. To measure the effect of pH on size and zeta potential, the nanoparticles were dispersed into phosphate buffer saline (PBS) of different pH (7.4, 6.5 and 5.0) and the particle size and zeta 6 ACS Paragon Plus Environment

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potential were measured after 10 min. All the measurements were done in triplicate. Fluorescent spectroscopy studies of free DOX and DOX-B6-SA-NP were performed at equivalent concentration of DOX to determine the successful loading of DOX in the nanoparticles using Fluorescence spectrophotometer (Agilent Cary Eclipse Fluorescence Spectrophotometer). The spectra in the range of 515 nm to 800 nm were collected at an excitation wavelength of 470 nm. The morphology of lipid nanoparticles (DOX-SA-NPs, DOX-B6-SA-NPs) was observed by atomic force microscopy (AFM, APCER, Italy) and transmission electron microscopy (TEM, TECNAI 200 Kv TEM, Fei, Electron Optics, USA) using the protocol in previously published reports

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. After physicochemical characterization loading content was determined by direct

method using HPLC. For determination of drug loading (DL), nanoparticles were isolated from 5 ml suspension by centrifugation at 40,000 rpm for 30 min using ultracentrifuge (Optima™ XPN, Beckman Coulter,Inc.,USA). The supernatant containing un-encapsulated drug was removed and the pellet was analyzed for drug content by HPLC. DL (%) were calculated according to the following formula: DL (%) = (Weight of DOX in particles/ Total weight of the particles) X 100 Physical stability and Stability in serum The long term physical stability of nanoparticles was measured by incubating the nanoparticle suspension in Phosphate buffer Saline (PBS,pH 7.4) for one month. Particle size and PDI was measured at regular intervals. Prototype formulation DOX-B6-SA-NP was also incubated in 0.2M NaCl and 10% FBS to determine the effect of ionic strength and serum proteins on stability of nanoparticles. Each study was conducted in triplicate. HPLC method development DOX concentrations in in-vitro release and in-vivo serum samples were analyzed using reverse phase –HPLC. The schimadzu HPLC equipped with 10 ATVP binary gradient pumps (Shimadzu) and RF-10AXL Schimadzu fluorescence detector was used for the analysis. C18 Lichrosphere Lichrocart column (5µm and 250 X 4.6 mm) was used for chromatographic separation using mobile phase composed of 25:75 ACN:Sodium acetate buffer (pH 3) at a flow rate of 1 ml/min. The injection volume was kept 40µl and the samples were analyzed under isocratic elution using fluorescence detector at excitation emission wavelength of 475 and 555 nm and the column temperature was kept constant 30 ºC. The developed method was further validated as per the guideline. 7 ACS Paragon Plus Environment


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Evaluation of pH on DOX release from nanoparticles The release pattern of DOX from nanoparticles was studied using dialysis bag method at 37ºC. The release media was consisting of phosphate-buffered saline (PBS) of different pH values (pH 5.8, pH 6.5, and pH 7.4). In brief the nanoparticle dispersion containing equivalent amount of DOX (2mg) was suspended in 1ml aqueous phase and transferred into a dialysis bag (MWCO 12KD). The dialysis bag was then immersed in 100mL PBS buffer of different pH with continuous stirring of 100 rpm at 37 ºC. At predetermined time points 200µl of release media was withdrawn and replaced with fresh 200 µl media. The amount of DOX in release sample was determined using HPLC with fluorescent detector. Cell culture Human breast cancer cell line MDA-MB 231was obtained from American Tissue Culture Collection (ATCC, Rockville, MD) whereas LA 7 cells are obtained from Indian Veterinary Research Institute (India). The cells were cultured in DMEM with 10% (v/v) Fetal Bovine Serum (FBS), 100 mg/mL streptomycin, 2mM glutamine, 1% NEAA and 100 U/mL penicillin in an incubator (Thermo Scientific, USA) at 37 °C under an atmosphere of 5% CO2 and 90% relative humidity. Cell uptake and intracellular release Confocal laser scanning microscopy (CLSM) The cell uptake and intracellular release behaviors form the developed Nanoparticles was determined by both CLSM and flow cytometry. For CLSM analysis MDA-MB 231 cells and LA 7 were seeded on poly-l-lysine coated coverslip in 6-well plates at a density of 1 x 103 cells per well in 1.5 mL of DMEM with 10 % FBS and incubated for 24 hr. After 24 hr the medium was replaced with media containing DOX-SA-NP and DOX-B6-SA-NPs at equivalent concentration of DOX (0.5µg/ml). After respective time period incubation cells were washed and fixed with 4% formaldehyde solution for 15 min at room temperature and then stained with DAPI. The cellular localization was visualized under a laser scanning confocal microscope (Olympus BX61242 FV1200-MPE, USA). The fluorescent intensity inside the cells was quantified using Image J software. Flow cytometry Quantitative determination of nanoparticles uptake by MDA-MB 231 and LA 7 cells was also was also performed by FACS analysis. Briefly, cells were seeded in a 6-well culture plate (5x105 8 ACS Paragon Plus Environment

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cells per well) and incubated with formulations as described above. After the respective time period incubation media was removed, cells were gently washed with PBS (pH 7.4) under dark conditions and then harvested and suspended in 0.5ml ml PBS. The samples were analyzed in flow cytometer (BD Biosciences, FACS Calibur). For FACS analysis all data of the mean fluorescence signal were obtained from a population of 10,000 cells. Cells without treatment were used as negative control groups. Endosomal escape MDA-MB-231 plated in 24-well plate containing poly-l-lysine coated cover slip (1 × 106 cells/well) and incubated for 24 hr. The culture medium was replaced with fresh culture medium containing FITC loaded B6-SA-NPs and cultured for 1 and 6 hr. After incubation the media was removed and fresh cell culture medium that contained Lysotracker red was added for another 1 hr. Finally the cells were gently washed with PBS, fixed with formaldehyde and then stained with DAPI (200ng/ml) for 15 min. The fluorescent images of the cells were taken by confocal microscope (Olympus BX61-242 FV1200-MPE, USA). In this study FITC was taken in place of DOX because “lysotracker red” used for lysosomes staining excite at the same wavelength as that of DOX. FITC loaded nanoparticles were prepared with similar protocol as mentioned previously except in place of DOX, FITC was used as fluorescent molecule. The amount of FITC in two groups was kept constant. Cytotoxicity The cytotoxicity of free DOX, DOX-SA-NPs and DOX-B6-SA-NPs on MDA-MB 231 and LA 7 cells was measured via MTT assay 28. In brief both the cells were separately seeded in 96-well plates at 3 x 103 cells per well in 100 µL DMEM medium containing 10% fetal bovine serum (FBS) and kept for overnight incubation at 37 ºC in 5% CO2. After this media was replaced and treated with free DOX, DOX-SA-NPs and DOX-B6-SA-NPs in 2% FBS containing DMEM media. The treatment was given at equivalent concentration of DOX for all the groups (ranging from 0.1 to 40µg/ml) for 24 hr. The treated cells were incubated at 37 ºC and then after respective time periods media was removed and treated with 20µl of MTT (4 mg/ml) for 4 h. After 4 hr 100µl of DMSO:IPA (1:1) was added to each well to dissolve the formed blue formazan crystals of live cells. The absorbance was measured on a microplate reader at 490 nm whereas cells with media only but without treatment were taken as control. All the experiments were repeated three times. 9 ACS Paragon Plus Environment


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Toxicity of blank nanoparticles was also measured on J774.3 cells to evaluate the biocompatibility of the of the developed lipid particles. Cell cycle analysis The effect of DOX on cell division of MDA-MB 231 cells was assessed using a Propidium Iodide (PI) -based assay

29

. Cells were plated in to six-well plates (5 × 105 cells/well) and kept

for 24 hr incubation at 37 °C to get the cells adhered. After 24 hr, the media was removed and cells were exposed to 1ml media containing different treatments of DOX equivalent to 2µg/ml. After the 24hr and 48 hr, DNA content of the cells were analyzed by FACS using previously published protocol

28

. In brief, after incubation the media was removed, cells were washed and

then harvested by centrifugation. The harvested cells were kept in 70% ethanol at 4°C for 2 hr and then suspended in 500µl PBS after washing. This cell suspension is then incubated with PI (10µg/ml) and RNAse (10µg/ml) at 4°C for 30 min in the dark and then analyzed for different stages of cell cycle using flow cytometer.

Apoptosis For apoptosis analysis cells treated with free DOX and different formulations containing the equivalent DOX concentration (2 µg/ml). At 24 hr post treatment, cells were harvested and processed by as per previously published protocol. In brief, after incubation cells were washed with PBS, harvested and resuspended in 500µl binding buffer in FACS tubes. The cell suspension in binding buffer was treated with DAPI (10 µl, 200 ng/ml) and Annexin-FITC (1.25 µl) and kept at 4ºC for 30 min under dark conditions to study early and late apoptosis. After this the cells were subjected to FACS. The experiments were done in triplicate. Cells without treatment were taken as control. Mitochondrial membrane potential Mitochondrial membrane potential (MMP) was measured using a JC-1 dye. MDA-MB 231 cells were seeded in 12-well plates (1 × 105 cells/well) and incubated overnight at 37°C and 5% CO2. Following this the media was removed and cells were incubated with fresh media containing different formulations at DOX equivalent 2 µg/ml for 24 hr. Subsequently, the cells were harvested, washed and resuspended in a 500 µL PBS containing JC-1 solution (15µg/ml). Cellassociated fluorescence was measured using flow cytometer (BD Biosciences, FACS Aria, Germany). 10 ACS Paragon Plus Environment

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In vivo Studies All the animals for in vivo experiments were taken from the national laboratory animal center (NLAC) and acclimatized for 1 weak under 12 hr day and night conditions. All the protocols for animal experiments were approved by the institutional animal ethical committee CSIR-CDRI.

Tumor induction in female SD rats Female SD rats of 3-4 weeks age (weight 50-60 gm) were selected to perform the pharmacokinetics and anti-tumor activity of the developed formulations. Animals selected for the experiments were housed for a week in 12 hr light and dark conditions prior to the LA-7 cells inoculation (Subcutaneous injection of 1X 105 cells / animal). After inoculation of cells to animals, regular observations of the animals were made. The studies were started when the tumor volume reached approximately 500 ±100mm3. Pharmacokinetics and bio-distribution Pharmacokinetic and bio-distribution studies were performed in tumor bearing female SD rats having tumor with volume (̴ 500 mm3). Rats were divided into three groups, each consisted of 5 subgroups (n=3 for each subgroups). Free DOX, DOX-SA-NPs and DOX-B6-SA-NPs were administered intravenously via tail vein at dose of 10 mg/kg 30-31. Blood samples were collected via oculi chorioideae using sparse sampling technique. Animals were grouped such that from each rat blood samples were collected for not more than two time points and after the second time point the rat was sacrificed for tissue collection. For each time point, a maximum of 500 µL was collected which was less than 10% of the circulating blood volume thereby precluding any chances of disturbing the normal physiological activity of rat. The blood samples were collected from retro orbital cavity and centrifuged to obtain the serum. The concentrations of DOX in samples were determined by the developed and validated HPLC methods. In brief 200 µL serum sample was precipitated using acidified 400 µL ACN: MEOH mixture, vortexed for 5 min and centrifuged at 8,000 rpm for 15 min and then 400µl of the supernatant was collected and dried in Maxi Dryer (Maxi Dry Plus, U.K laboratories). The dried sample was then dissolved in the mobile phase for HPLC analysis. For analysis of drug in organs, the organs were homogenized in PBS to form a tissue homogenate and this tissue homogenate was extracted with similar protocol as for serum.



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In vivo tumor regression study For tumor regression study animals were divided into four groups with 6 animals in each group. The animals were observed daily and when the tumor volume reached approximately 500 mm3, they were injected with i.v route free DOX, DOX-SA-NPs and DOX-B6-SA-NPs at equivalent to 10mg/kg DOX from the tail vein. The formulations were administered after every 4 th day until the three doses. The control group was also maintained with administration of only Physiological saline. Tumor growth was checked every fourth day. The tumor volume was calculated as by using the following formula Volume (V) =1/2* L*W2, where W and L denote the short and long diameters of the tumor tissue. Rats were sacrificed after the 30th day. The body weight was measured continuously as the indicator of general toxicity. Toxicity studies Survival analysis Similar to the tumor regression study animals were divided into four groups with 6 animals in each group. The animals were observed daily and when the tumor volume reached approximately 500 mm3, animals were treated as per the protocol described above for in vivo antitumor growth experiment. However unlike regression study which was of 20days survival study was conducted upto 40 days after the treatment started. The survival rate of animals in different groups was also measured during this period and analyzed using graph pad prism 5.0 software.

Hemolysis assay Hemolytic activity of the developed lipid nanoparticles was evaluated as per the previous protocol with slight modification 32. Briefly fresh blood from rat was collected and centrifuges to obtain the red blood cells. The RBC were washed and suspended in PBS. This RBC suspension was incubated with different concentration of formulation for 12 hrs in incubator at 37 ºC where PBS was taken as negative control whereas treatment of Triton X was taken as positive control. After incubation the suspension was centrifuged to separate the non-lysed RBCs and supernatant was analyzed for free hemoglobin content due to RBC lysis. The % hemolysis was calculated using the formula: Hemolysis (%) = (A sample – A negative control)/(A positive control- A negative control)*100, Histology


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The rats were sacrificed at the end of the tumor regression study and the major organs (heart, liver, spleen, lung and kidney) were collected, fixed in 10% paraformaldehyde (PBS buffered) overnight, and then small pieces of fixed organ were embedded in wax blocks. About 5 µm thick sections were obtained using microtome (Leica) and subjected to hematoxylin and eosin (H&E) staining to visualize histological changes in their respective organs27 . Immunogenicity Immunogenicity of nanoparticles was examined in rats. The mice were injected with DOX-SANP and DOX-B6-SA-NP at equivalent dose of 10mg/kg DOX and blood samples were collected post 10 hr and 24 hr. Serum was separated from blood by centrifuging at 800 rpm for 15 min and then the cytokines levels IL-6, TNF-α and IL-1β were determined using Duoset ELISA kits (R&D System, San Diego, CA, USA) as per the manufacturer's protocol.

Statistics Student t-test was used for the significance analysis where in most of the cases the values (p

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