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Click biotinylation of PLGA template for biotin receptor oriented delivery of doxorubicin hydrochloride in 4T1 cell induced breast cancer Yuvraj Singh, K K Durga Rao Viswanadham, Arun Kumar Jajoriya, Jayagopal Meher, Kavit Raval, Swati Jaiswal, Jayant Dewangan, H. K. Bora, Srikanta Kumar Rath, Jawahar Lal, Durga Prasad Mishra, and Manish Kumar Chourasia Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00310 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 22, 2017
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Molecular Pharmaceutics
Click biotinylation of PLGA template for biotin receptor oriented delivery of doxorubicin hydrochloride in 4T1 cell induced breast cancer Yuvraj Singha, K.K.Durga Rao Viswanadhama#, Arun Kumar Jajoriyab, Jayagopal Mehera, Kavit Raval a , Swati Jaiswalc, Jayant Dewangand, H. K. Borae, Srikanta Kumar Rathd, Jawahar Lalc, Durga Prasad Mishrab, Manish Kumar Chourasiaa* a
Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow- 226031, India
b
Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow- 226031, India
c
Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow226031, India d
Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow- 226031, India
e
Laboratory animals facility, CSIR-Central Drug Research Institute, Lucknow- 226031, India
#
Current affiliation: Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver,
British Columbia V6T 1Z3, Canada.
* Corresponding Author Manish K. Chourasia Sr. Scientist Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, India, 226031 Ph. No: +91 522-2772450, 2772550. Fax: +91 522 2623405 Email:
[email protected] CSIR CDRI communication XXX-XXX
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Abstract PLGA was functionalized with PEG and biotin using click chemistry to generate a biotin receptor targeted copolymer (Biotinylated-PEG-PLGA) which in turn was used to fabricate ultrafine nanoparticles (BPNP) of doxorubicin hydrochloride (DOX) for effective delivery in 4T1 cell induced breast cancer. However, adequate entrapment of a hydrophilic bioactive like DOX in a hydrophobic polymer system made of PLGA is not usually possible. We therefore modified a conventional W/O/W emulsion method by utilizing NH4Cl in the external phase to constrain DOX in dissolved polymer phase by supressing DOX’s inherent aqueous solubility as per common ion effect. This resulted in over eight fold enhancement in entrapment efficiency of DOX inside BPNP, which otherwise is highly susceptible to leakage due to its relatively high aqueous solubility. TEM and DLS established BPNP to be sized below 100 nm, storage stability studies showed that BPNP were stable for one month at 4°C, and in vitro release suggested significant control in drug release. Extensive in vitro and in vivo studies were conducted to propound anticancer and antiproliferative activity of BPNP. Plasma and tissue distribution study supplemented by pertinent in vivo fluorescence imaging mapped the exact fate of DOX contained inside BPNP once it was administered intravenously. A comparative safety profile via acute toxicity studies in mice was also generated to out rightly establish usefulness of BPNP. Results suggest that BPNP substantially enhance anticancer activity of DOX whilst simultaneously mitigating its toxic potential due to altered spatial and temporal presentation of drug and consequently deserve further allometric iteration. Keywords: nanoparticles, common ion effect, PLGA, doxorubicin hydrochloride, click chemistry, active targeting
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Molecular Pharmaceutics
INTRODUCTION Doxorubicin hydrochloride (DOX) is a drug of choice in variety of cancerous ailments. DOX along with adjuvants is recommended as a first line anti-cancer drug in NCCN’s 2016 breast cancer guidelines1. It interferes in DNA synthesis by binding to topoisomerase II, damages preexisting DNA via intercalation, generates reactive oxygen species, induces mitochondrial membrane depolarization by releasing cytochrome c and obliterates membranous integrity of treated cells2. When used as a mono-therapeutic option, after relapse or failure of combination therapy, DOX induces response in 40 % first line cases and 20% second-line cases. Despite these attributes, DOX is connected to numerous side effects (dose-dependent cardiotoxicity, nausea, myelotoxicity, gastrointestinal shedding, and acute cramps) which restrict its utility in a palliative environment3. DOX consists of tetracycline quinoid aglycone doxorubicinone and amino sugar (daunosamine) linked together by a glycosidic linkage (Figure 1), which is extremely susceptible to acidic degradation prevalent in gastro intestinal tract4.
Figure 1: Chemical structure of DOX. Equilibrium between salt form and ionized form of drug is rapidly attained in water, yielding to solubilisation.
This attribute rules out oral administration and necessitates usage of higher parenteral dose which inadvertently contributes towards cardiac toxicity. DOX is usually administered intravenously as a hydrochloride salt and therein lays a major delivery related stumbling block. It is inconceivable to adequately entrap a hydrophilic moiety (aqueous solubility of DOX is 10 mg/mL) in a hydrophobic polymer like PLGA or its derivative as drug is subject to leakage in surrounding media during aqueous processing of formulation (Figure 2C). Although we along with others have attempted to use base form of DOX to ensure its entrapment, it all adds to tediousness of formulation development causing quantifiable drug loss as the process of converting DOX to its base form is inefficient5. To circumvent such inefficient techniques we have utilized principle of common ion effect which was theoretically expounded during the last century itself, but has not been utilized anywhere in pharmaceutics. We try to increase entrapment of a highly water soluble salt form of a drug (i.e. DOX) in a hydrophobic polymer nanoparticle system (derived from PLGA), despite drug’s tendency to leach out, by supressing its aqueous solubility via introduction of a stronger electrolyte (NH4Cl) which bears a chloride ion common to DOX in the external phase of formulation. The proposed strategy if successful could work for other bioactives as well (because most drugs lay in the weak electrolyte category) and potentially open up a new methodology of entrapping hydrophilic drugs in polymeric matrices. Selection of PLGA as a delivery construct for DOX was based on PLGA’s proven industrial and commercial utility (Zoladex®, Lupron®, Sandostatin®, AtriDox®, Buduperon®, and so on). Further advantages of PLGA can be gauged from its biodegradability, reproducible fabricability and sheer number of biomedical devices/formulations undergoing advanced stages of clinical trials6. PLGA is famed for producing ultra-fine spherical particles7 and the bi product it generates enter carboxylic acid cycle, thereby undergoing innocuous elimination or reutilization in bodily metabolomics8.
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Figure 2: Work in brief. (A) Reaction scheme for synthesis of biotinylated-PEG-PLGA using click chemistry. (B) Biotinylated-PEG-PLGA was employed to generate nanoparticles of DOX via W/O/W emulsion method. Using a strong electrolyte like NH4Cl bearing a common ion to doxorubicin hydrochloride in the external aqueous phase supresses aqueous solubility of DOX and traps it in organic phase containing Biotinylated-PEG-PLGA. (D) Once polymer solvent vaporizes, biotin tagged PLGA nanoparticles with multifold higher DOX content are obtained which when tested in vivo are expected to elicit specific cytotoxic action against biotin receptor overexpressive 4T1 breast cancer cells. Key: 1a (Propargyl alcohol); 2a (PLGA); 3a (Clickable alkyne ended PLGA); 4a (Biotin-PEG-Azide); 5a (Biotinylated-PEGPLGA); DCC (N,N'-Dicyclohexylcarbodiimide); DMAP (4-Dimethylaminopyridine); tBuOH (tertiary butyl alcohol); r.t (room temperature).
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Molecular Pharmaceutics
Although annals of nanotechnology assisted anticancer delivery are heavily interspersed with studies where researchers have exploited minute dimensions of PLGA nanoparticles to passively target tumor cells, it is generally agreed that specific targeting with limited/no off target effects as postulated by Ehlrich in ‘magic bullet’ concept is only realizable via active targeting9. We consequently decorated PLGA with biotinylated-PEG by utilizing click chemistry to generate a cancer cell homing copolymer: biotinylated-PEG-PLGA. Presence of PEG in template was expected to accord nanoparticles long circulation half-lives, which in turn would assist in their probability of reaching tumor microenvironment10 for targeted delivery of potent cytotoxic DOX in 4T1 cell induced allogenic breast cancer model11. It is assumed, these nanoparticles after accumulation at intended destination owing to their size, would then be preferentially internalized by cancer cells due to specific interaction of their biotin component with overexpressed biotin receptors on 4T1 cells12. In the direction of scheme depicted in Figure 2, nanoparticles made of biotinylated-PEG-PLGA containing high load of doxorubicin hydrochloride (BPNP) were prepared via a common ion effect modified W/O/W emulsion method. Extensive studies were conducted to propound anticancer activity of developed nanoparticles in 4T1 breast cancer cells. Pharmacokinetic intonation in DOX brought about by its loading in nanoparticles was observed by both LC-MS and in vivo imaging. A comparative safety profile via acute toxicity studies in mice was also generated. In vivo anticancer activity in 4T1 cell induced breast cancer carrying Balb/C mice model was done to outrightly establish utility of nanoparticles. EXPERIMENTAL Material Doxorubicin hydrochloride (DOX) was received as generous gift from Fresenius Kabi, Gurgaon, India. Poly lactide-co-glycolide (PLGA) with molecular weight 50000 Da (50:50 grade) was bought from DURECT, Birmingham, AL, USA. Pluronic 127 (PF 127), Biotin-PEG-Azide conjugate, propargyl alcohol, copper sulfate, sodium ascorbate, fetal bovine serum (FBS), antibiotic solution (penicillin/streptomycin, 0.1% v/v), dialysis membrane (10000-12000Da), ribonuclease A, and RPMI 1640 Medium, 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT), were purchased from Sigma Aldrich, St. Louis, MO, USA. 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI), JC-1 Dye were procured from Invitrogen, OR, USA. Three-stage purified water was obtained from Milli-Q purifier (Millipore, Milli-Q plus 185, Bedford, MA, USA). All solvents used were of HPLC or LC-MS grade. The chemicals were used as provided. Solubility studies Excess of DOX was added to 5ml water or fixed strength aqueous solutions of different electrolytes (as mentioned in Table 1) in 10 mL capped vials. The dispersions so formed were shaken on a bench top orbital shaker (Thermo Fischer, India) at room temperature (25±2°C) for a period of 24 h. A fixed aliquot from the supernatant of each vial was diluted appropriately using water and subjected to chromatographic quantification (detailed in supplementary data). Solubility experiments were conducted in triplicate. Table 1: Aqueous solubility of doxorubicin hydrochloride in presence of different chloride salts NaCl (Ki 0.0047) KCl (Ki 0.0059) NH4Cl (Ki 0.0092)
ZnCl2 (Ki 0.0259)
NaCl (mg/mL)
DOX solubility (mg/mL) in presence of NaCl
KCl (mg/mL)
DOX solubility (mg/mL) in presence of KCl
NH4Cl (mg/mL)
DOX solubility(mg/ mL) in presence of NH4Cl
ZnCl2 (mg/mL)
DOX solubility (mg/mL) in presence of ZnCl2
0
10±0.5
0
10±0.5
0
10±0.5
0
10±0.5
2
8.4±0.3
2
8.1±0.3
2
6.0±0.2
2
3.9±0.3
3
7.8±0.2
3
7.5±0.5
3
3.7±0.3
3
3.1±0.5
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5
7.3±0.3
5
7.4±0.4
5
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2.6±0.6
5
2.7±0.1
Development of Biotinylated-PEG-PLGA via click chemistry Synthesis of Propargyl PLGA Conjugate (3a) A mixture of PLGA (2a) (1 gm, 0.02 mM, 1 eq.), propargyl alcohol (1a) (0.6 mM, 3eq.), and N,Ndimethylaminopyridine (DMAP) (0.06 mM 0.3 eq.) was dissolved in 50 mL dry DCM in a 100 mL round-bottom flask and stirred in ice bath for half an hour. To this mixture, DCC (1 eq.) dissolved in 15 mL dry DCM was added, and the sample was stirred in an ice bath for 1 h. The reaction mixture was further stirred for 24 h at room temperature. Reaction was monitored by IR analysis. After completion, reaction mixture was filtered. Filtrate was washed with water (2x40 mL) and brine (1x40 mL), dried over anhydrous Na2SO4, and evaporated. After removal of solvent under vacuum, extract was washed with ether and recrystallized with ethyl acetate and hexane (1:2) to get clickable propargyl PLGA (3a); 85% yield. Synthesis of Biotinylated-PEG- PLGA via click reaction Propargyl PLGA conjugate 3a (0.500 mg, 009 mM, 1 eq.) was dissolved in tBuOH–H2O (1 : 1, 10 mL) by stirring for 15 min. Sodium ascorbate ( 0.0029 mM, 0.3 eq.) was then added and allowed to stir for 15 min followed by copper (II) sulfate ( 0.0027 mM 0.1 eq.). After 15 min Biotin-PEG-Azide (4a) (0.01 mM, 1 eq.) was added to the mixture and it was stirred at room temperature in nitrogen for 24 h. The reaction conversion was confirmed by IR analysis. Upon completion, the mixture was evaporated under vacuum. The crude resultant was diluted with water and extracted with DCM (2×20 mL). The extract was washed with brine solution and dried over anhydrous sodium sulphate (Na2SO4). After removal of the solvent under vacuum, extract was washed with ether three times. It was then precipitated in DCM and excess ether at low temperature to yield final compound with 80 % yield. The final compound, 5a, (Biotinylated-PEG-PLGA) was confirmed by 1H NMR. Preparation of nanoparticles Biotinylated-PEG-PLGA nanoparticles containing DOX (BPNP) were prepared by a modified W/O/W double emulsion solvent diffusion technique with minor changes for trapping hydrophilic molecule DOX13. Briefly, as per Table 2, a specified quantity of polymer (PLGA-PEG-Biotin) and PF127 (3% w/v) were dissolved in 3 mL ethyl acetate. To this organic solution, 1 mL triple distilled water containing known amount of drug was added. The mixture was then sonicated over an ice bath using a probe sonicator (Sonics, USA) at 40 % amplitude for 3 min to form a W/O emulsion. The resulting primary emulsion was added to 4 mL aqueous NH4Cl solution (5mg/mL), and re-sonicated over an ice bath at 30% amplitude for 10 min to form a W/O/W double emulsion. Ethyl acetate was eliminated by evaporation under reduced pressure, causing hardening of polymer around drug to form fully ripened nanoparticles (BPNP). Nanoparticle suspension was dialysed against triple distilled water to remove free drug and dissolved NH4Cl. Optimization studies were conducted to estimate effect of formulation and processing variables on size and entrapment efficiency of BPNP. For comparative purposes, blank nanoparticles were similarly developed (PNP or NP) without any drug. The formulated nanoparticles were stored in cold refrigerated conditions until further use. The final coded formulations were as follows: Biotinylated-PEG-PLGA nanoparticles of DOX (BPNP); PLGA nanoparticles of DOX (DPNP); Blank/dummy Biotinylated-PEG-PLGA nanoparticles (NP); Blank PLGA nanoparticles (PNP).
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Table 2: Batch details of BPNP with selected physicochemical characters
Batch
Polymer (mg)
Drug Polymer Ratio
Particle Size (nm)
PDI
ZP (mV)
%EE
F1
10
~1:1
48±4
0.18±0.07
-6.1 ±2.1
3.1±1.3
F2
15
2:3
72 ±3
0.22±0.11
-7.8±2.0
5.2±1.5
F3
30
1:3
81±3
0.18±0.05
-3.9±0.9
7.4±2.8
F4
40
1:4
98±4
0.14±0.07
-3.7±1.3
12.0±1.6
F5
60
1:6
155±5
0.23± 0.14
-8.4±2.7
14.5±2.2
Physicochemical characterization Particle size and polydispersity index were measured by dynamic light scattering. Zeta potential was evaluated by calculating mean electrophoretic mobility of particles. Formulations were appropriately diluted and analyzed at ambient temperature using Malvern zetasizer (Nano ZS, Malvern Instruments, UK)14. Entrapment efficiency was determined by employing reverse phase liquid chromatography. Briefly, specified volume of formulation was dissolved in 1:1 mixture of DMSO and acetone, and vortexed for 5 min. The mixture was thereafter diluted with water and analyzed on a HPLC. Optimized batch was also subjected to storage stability studies at room temperature (25±2 °C) and cold refrigerated conditions (4°C). Samples were drawn on fixed days and observed for increment/decrement in particle size and entrapment efficiency15. DSC analysis was performed on synthesized copolymer biotinylated–PEG-PLGA, dummy nanoparticles made out of biotinylated–PEG-PLGA, doxorubicin hydrochloride, and BPNP using a differential scanning calorimeter (PerkinElmer, USA). Dried samples (lyophilized or otherwise) weighing approximately 5 mg, were placed in hermetically sealed aluminum pans and heated at uniform rate of 10°C/min up to 800°C in an inert environment maintained under dynamic nitrogen purging. Shape, size and surface appearance of nanoparticles was analysed by electron microscopy (TEM). Briefly, diluted BPNP suspension was smeared onto a 300-mesh copper grid in form of a film. The grid was subsequently coated with 2% w/v uranyl acetate. Excess solution was blotted off and the grid was dried using liquid nitrogen. Dried BPNP were observed at 80 kV by using TECHNAI G2 20 S-TWIN (FEI Netherlands) instrument and photomicrographs of different fields at several appropriate magnifications were taken. Dissolution study was conducted using a dialysis tube. BPNP, DOX solution and DPNP with equivalent DOX content (2 mg) were sealed in an activated dialysis bag and submerged in 50 ml phosphate buffer saline (PBS pH 7.4). The dissolution medium was magnetically stirred at 100 rpm and temperature was maintained within 0.5°C of 37°C. Periodic sampling was done and amount of drug released was analyzed by reverse phase HPLC. Obtained dissolution profile was fitted in different release models via linear regression method. This was done to excavate probable mechanistics of drug release. Quantitative and qualitative cell uptake study: dependency on biotin receptors Flow cytometry was used to quantitatively assess cellular uptake of DOX loaded nanoparticles (BPNP vs. DPNP vs. DOX solution). Briefly, 4T1 cells were seeded in six well plates at a density of 0.5x105 cells per well for plate adhesion and priming. Subsequently selected wells were incubated with media containing BPNP, DPNP and DOX solution for 6 hours followed by washing with PBS. Cells were then trypsinized, pelleted, resuspended in PBS and analyzed for fluorescence produced by internalized DOX by using flow cytometer (FACS Calibur, software cell quest Pro). To establish dependency of BPNP on biotin receptor mediated uptake, an additional well of cells was first treated with BiotinACS Paragon Plus Environment
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Blocking Kit (E-21390, Thermo-Fischer Scientific, India) as per manufacturers protocol to saturate the surface expressed biotin receptors and subsequently subjected to BPNP for 6 hours and analysed as above by FACS. For fluorescence microscopy, 4T1 cells were seeded in 6-well plates containing poly-l-lysine coated coverslips for 24 h. Thereafter cells were treated with DOX solution, DPNP and BPNP for 6 hours. We also analysed time dependent uptake of BPNP. After staining, cells were rinsed repeatedly in PBS and re-incubated with formaldehyde (4% v/v in PBS) at 37 °C for 15 min. The fixed cells were then aspirated. The coverslip left behind, containing treated cells was mounted on slides using ProLong® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, India) and observed under a fluorescence microscope at 60X (Nikon Eclipse TS100 with fluorescence attachment Nikon-CHGFI). Cytotoxicity study and in vitro hemolysis MTT-based in vitro assay was performed to compare cytotoxic effects of different groups on 4T1 cells. Briefly, cells (0.5×104 cells per well) in 96-well culture plates were seeded overnight in a controlled environment of 37 °C, 5% CO2. Cells were then treated with DOX solution, BPNP, DPNP, PNP and NP dispersed in media equivalent to different concentrations of DOX. After 24 h, the treatment media from wells was aspirated off and replaced with fresh media containing MTT solution (0.5 mg/mL) for 4 h. The formazan crystals so formed were dissolved by adding 0.2 mL of DMSO to each well and absorbance of resulting solution was measured at 570 nm using a multiwell scanning spectrophotometer (PowerWave XS, Biotek, VT, USA). Nonlinear regression analysis was applied to determine inhibitory concentration 50 % (IC50) for each treatment group16. Hemo compatibility of nanoparticles was evaluated in erythrocytes isolated from blood of SD rats by centrifugation (1000×g for 10 min). The settled RBCs were suspended in PBS. Accurately measured 2mL RBC suspension was mixed with aliquots of drug and formulation in triplicate to attain a final DOX concentration of 0.5 to 25 (µg /ml). RBCs dispersed in PBS only and PBS containing 10% Triton- X were used as negative and positive control respectively. The RBC-drug aliquots were incubated at 37 ºC in an atmosphere of 5% CO2 for 2 h. After incubation, samples were centrifuged at 800xg for 10 min, and supernatant was collected in 96 well plates to be analyzed spectrophotometrically at 540 nm. Cell cycle distribution Propidium iodide (PI) was used as a fluorescent marker for cell cycle analysis. 4T1 cells were grown in six well plates (106cells/well) and allowed to adhere. Free DOX, BPNP and DPNP (each equivalent to 50 nM DOX respectively) were added into different wells for a period of 24 hours. After stipulated time, cells were detached, rinsed in PBS and fixed in chilled ethanol (70% v/v). Thereafter cells were spun, pelleted, resuspended in 250µL PBS and treated with ribonuclease A (100 µg/mL) for 15 min followed by staining with PI (50 µg/mL) for another 30 min. Fluorescence induced by association of PI and remnant genetic material of permeabilized cells was measured employing a flowcytometer (BD Biosciences, FACS Aria, Germany) and translated into the phase of cell cycle arrest induced by different treatments. Apoptosis To determine extent and type of apoptosis induced by DOX solution, BPNP and DPNP we utilized Annexin V and PI kit (Invitrogen, CA, USA). Protocol prescribed in the kit was strictly adhered to and 4T1 cells (cultured as in cell uptake) were treated with different formulations (all equivalent to 100 nM DOX) for 24 h. Subsequently, cells were trypsinized, spun, washed thrice with PBS and dispersed in 500 µL binding buffer. Thereafter, cells were incubated for 30 min in dark after adding annexin VACS Paragon Plus Environment
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FITC (5 µL) and PI (5 µL). Finally these cells were subjected to flow cytometry so as to differentiate their staining pattern.
In vivo antitumor activity 5-7 week old female Balb/C mice weighing 15-25 g acted as model for breast cancer. Murine mammary carcinoma cell line (4T1) was injected subcutaneously into fat pad of mammary gland in lower right quadrant of mice’s abdomen (1×106 cells/mice). After a waiting period of 10 days post inoculation (tumors became palpable from 7 days on), a careful screening was done to select animals with adequate tumor volume (100-150 mm3). The selected animals were divided into six groups of five each (n=5). A dosing interval of 72 hours was adapted with DOX solution, BPNP and DPNP administered via intravenous entry into mice’s tail on day 0 (day of treatment initiation), 3, 6 and 9. Another group was dosed similarly with PBS to form the control group. The study was ended on 27th day by sacrificing animals and harvesting their tumors for subsequent processing [sliced into thin sections (5-8µm) and embedded in paraffin] which included haemotoxyline and eosin staining, immune-staining for PCNA and detection of apoptotic cells population through a dead end fluorometric TUNEL assay using biotin-dUTP (biotinylated deoxyuridine riphosphate) and avidinFITC as an apoptotic marker (TUNEL apoptosis detection kit, Millipore). The purported efficacy of administered treatments was gauged by measuring tumor dimensions during the study as well as on the day of termination. A digital Vernier’s caliper was employed to do the same. Pharmacokinetics Pharmacokinetic study of DOX and its formulations was carried out in 4T1 cell induced tumor bearing Balb/C mice. DOX solution, BPNP and DPNP were administered intravenously to conscious mice (via the caudal vein) at a dose of 4 mg/kg. Blood (approximately 100 µl) was collected by puncturing retero-orbital plexus with heparinised capillaries at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 24, 48 and 72 h post dosing. Sparse sampling was applied for blood collection and the total volume of blood collected from each mice was not more than 3% of total body volume. Blood samples were centrifuged at 5000 rpm for 10 min at 4°C and plasma was separated into clean and neatly labelled tubes. All samples were stored at -80°C until analysis. A different set of animals which had received the same treatment as above were sacrificed at 2, 6, 10, 24, 30 and 72 h post dosing and organs such as spleen, kidney, heart, and tumor were collected. Protein precipitation approach was employed for sample clean-up. To the plasma sample or tissue homogenate (30µl; blank, spiked or test), 170 µL acetonitrile containing 30 % ammonium acetate buffer (pH 3.5, 0.01M) was added. The samples were vortex-mixed for 5 min followed by centrifugation for 10 min at 10,000 rpm. Clear supernatant (80 µl) was transferred into HPLC vials and 20 µl was injected into LC-MS/MS system (refer to supplementary data). Win Nonlin 6.0 (Pharsight Co., Mountain View, CA, USA) was utilized to determine various pharmacokinetic parameters.
In vivo fluorescent imaging To complement pharmacokinetic studies, distribution and localization of DOX was measured by using live in vivo imaging system IVIS® Spectrum (Caliper Life Sciences/ PerkinElmer). Non-invasive whole body fluorescent images of DOX solution, BPNP and DPNP (equivalent to 4 mg/kg DOX) treated mice (bearing 4T cell inuced tumor) were generated by subjecting them to frontal examination at 535 nm. Subsequently generated emission was recorded at 560 nm. Images were acquired for 1 minute at pre fixed time points (5 min, 6 hours and 24 hours after dosing) and emission emanating from mice was analysed using Living Image® Software 4.4. In order to facilitate accurate imaging, mice were pre-anaesthetized using ketamine (20 mg/kg i.p.) before their placement inside imaging chamber of instrument.
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Toxicity study Acute toxicity study was performed on six week old Balb/C mice (weighing ~ 20 g) according to institutionally approved protocol. Mice were divided into four groups of five animals each. The first group acted as control and was treated with PBS via tail vein whereas the second, third and fourth group received DOX solution, DPNP and BPNP respectively at a dose equivalent to 10 mg/kg DOX. This dosing cycle was repeated thrice, with 72 h between each dose. After 14 days, all animal were sacrificed and major organs such as liver, heart, and kidney were harvested. For histological examination excised organs were fixed in formalin (10% in normal saline), rooted in paraffin blocks, sliced to very thin sections using automatic micro tome (Leica, model- 2155) and stained with haemotoxyline and eosin. The sections were observed under an optical microscope to grossly map their cellular architecture (Eclipse 50i, Nikon, Japan). For biomarker assay, heart of each animal was purged in liquid nitrogen at the moment of excision, thereafter cut into weighted pieces and homogenized in PBS (weight to volume ratio of 1:5) at 20000 RPM for 3 min using an ULTRA TURRAX® T-10 basic (Ika, Germany). The tissue homogenate was used for estimation of catalase (CAT), malondialdehyde (MDA) and superoxide dismutase (SOD). All three enzymes were estimated as per protocols prescribed in their respective estimation kits (Sigma Aldrich, St. Louis, MO, USA) Statistical evaluation Statistical appraisal of stability batches was carried out by applying one way analysis of variance (ANOVA) followed by Dunnett's Multiple Comparison Test using readings of day 1 as control. Results of in vivo efficacy studies were compared by two way ANOVA followed by Bon-Feroni post-test. Point to point comparison required in experiments such as IC50 were done with unpaired t-tests. p
