Multifunctional Glycoconjugate Assisted Nanocrystalline Drug Delivery

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Biological and Medical Applications of Materials and Interfaces

Multifunctional Glycoconjugate Assisted Nanocrystalline Drug Delivery for Tumor Targeting and Permeabilization of Lysosomal-Mitochondrial Membrane Gitu Pandey, Naresh Mittapelly, Venkatesh Teja Banala, and Prabhat Ranjan Mishra ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18699 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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ACS Applied Materials & Interfaces

Multifunctional Glycoconjugate Assisted Nanocrystalline Drug Delivery for Tumor Targeting and Permeabilization of Lysosomal-Mitochondrial Membrane Gitu Pandey †‡, Naresh Mittapelly†‡, Venkatesh Teja Banala†, Prabhat Ranjan Mishra†‡*

† Pharmaceutics and Pharmacokinetics Division, CSIR- Central Drug Research Institute ‡ Academy of Scientific and Innovative Research, Chennai, India.

* Pharmaceutics and Pharmacokinetics Division, PCS 002/011 CSIR-Central Drug Research Institute B.S. 10/1, Sector-10, Jankipuram Extension, Lucknow E. mail: [email protected]; [email protected]

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Abstract Nanotechnology has emerged as the most successful strategy for targeting the drug payloads to the tumors with potential to overcome the problems of low concentration at the target site, nonspecific distribution and untoward toxicities. Here, we synthesized a novel polymeric conjugate comprising of chondroitin sulfate A and polyethylene glycol using carbodiimide chemistry. We further employed this glycoconjugate possessing the propensity to provide stability, stealth effect and tumor targeting via CD44 receptors, all in one, to develop a nanocrystalline system of docetaxel (DTX@CSA-NCs) with size< 200nm, negative zeta potential and 98% drug content. Taking advantage of the enhanced permeability and retention effect coupled with receptor mediated endocytosis, the DTX@CSA-NCs cross the peripheral tumor barrier and penetrate deeper into the cells of tumor mass. In MDA-MB-231 cells, this enhanced cellular uptake was observed to exhibit higher degree of cytotoxicity and arrest in G2 phase in a time dependent fashion. Acting via mitochondrial-lysosomotropic pathway DTX@CSA-NCs disrupted the membrane potential and integrity and outperformed the clinically used formulation.

Upon

intravenous

administration,

the

DTX@CSA-NCs

showed

better

pharmacokinetic profile and excellent 4T1 induced tumor inhibition with significantly less off target toxicity. Thus, this glycoconjugate stabilized nanocrystalline formulation has the potential to take nano-oncology a step forward. Keywords: Docetaxel, CD44 targeting, covalent conjugation, fixed aqueous layer thickness, lysosomal membrane integrity, mitochondrial membrane potential, 4T1 breast tumor


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1. Introduction In recent years, nanotechnologically engineered drug delivery systems have been extensively employed for achieving the better therapeutic outcome in nanomedicine especially in cancer. They have emerged as the most successful strategy for selective delivery of drugs to tumors with high payload and circumventing the undesired reactions. The angiogenesis and aberrant tumor vasculature makes passive targeting via the enhanced permeability and retention effect (EPR) an interesting approach.1 However, this passive strategy alone is not adequate for tumor targeting because a large number of administered nanoparticles still accumulate in vital organs like liver, lungs and spleen. Also the extravasations following tumor uptake and poor tumor penetration contribute towards lower ‘on site’ concentration.2 Considering this, a receptorligand based active targeting coupled with a modality for long term blood circulation will be more suitable for achieving an enhanced tumor accumulation, distribution within tumor and reducing untoward adverse effects. The success of this approach depends upon the over expression of distinct type of proteins, called receptors, on the tumor cell surface as compared to normal somatic cells. The receptors like human epidermal growth factor receptors (HER-2), estrogen receptors (ER), progesterone receptors (PR), folate receptors (FR), cluster designation 44 receptors (CD44) etc. have been exploited for the above said purpose.3-4

5

Among these

receptors, the CD44, a multifunctional transmembrane glycoprotein, is attracting considerable research interest owing to its illustrious role in cancer stem cell maintenance, tumor invasion, migration, metastasis and chemoresistance.

6-7

It is also being used for actively targeting the

nanocarriers to the tumors for theranostic purpose using hyaluronic acid and chondroitin sulfate as targeting ligand.8-9 3 ACS Paragon Plus Environment


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Chondroitin sulfate is an anionic gylcosaminoglycan consisting of unbranched polysacchride chains of variable length. It is a biocompatible polymer and has proven favourable action in angiogenesis, tumor invasion and metastasis.10 A vast amount of research has been done in order to study the role of chondroitin sulfate in targeting the cancerous cells. To mention a few, Vargheese et al prepared theranostic chondroitin sulfate nanoparticles coupled with fluorescein exploiting the pi-pi interaction for stabilization and targeting

10

and Hu et al formulated

chitosan - chondroitin nanoparticles bearing doxorubicin for effective CD44 targeting and reported enhanced uptake and improved in vivo efficacy. 11 Different type of nanoparticulate systems have been employed for the delivery of the payload to the desired site, reducing the off target toxicity and for altering physicochemical properties in favorable direction.12-13

14

These include polymeric nanoparticles, micelles, lipidic

nanoparticles, nanocapsules, and nanocrystals etc. Among these nanocarriers, the nanocrystals possess some specific advantages like ultra high mass fraction of therapeutics (theoretically hundred percent), minimal involvement of excipients, acceptability for both oral and intravenous administration, ease of size tailoring and better industrial scalability.15 They increase the solubility and dissolution velocity following the Ostwald- Freundlich and NoyesWhitney equation. Moreover, they offer the possibility of surface modification with targeting ligand of interest, and are suitable for a large number of drugs across the therapeutic categories.16 Owing to these virtues, nanocrystals have been explicitly employed and have shown exciting results with various anticancer agents like taxanes, camptothecin, hydroxycamptothecin, bicalutamide, melarsoprol etc. As targeted nanocrystals, like any other nanocarriers system, reach their targeting site only after blood circulation and extravasation, 4 ACS Paragon Plus Environment

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the retention of the delivery vehicle in the systemic circulation for a prolonged duration is a prerequisite. Here comes the role of pegylation which imparts the stealth properties to the nanocarriers and hence prevents the protein adsorption (protein corona), opsonization and quick systemic clearance. 17 This long circulating nanocrystalline system could be very well applied to Docetaxel (DTX), a fundamentally important drug which acts via hyperstabilization of microtubule by targeting β subunit of the tubulin heterodimer and suppressing spindle microtubule dynamics. Clinically, it has demonstrated more potent antitumoral activity than paclitaxel and lacks cross resistance with it. Despite having proven clinical superiority over many other anticancer drugs, some inherent physicochemical properties like low aqueous solubility and proclivity towards P-gp efflux pump renders it unsuitable for administration as a purely water based solution and necessitates the administration with polysorbate 80 (PS 80) based solution. This adds to the woes as PS 80 itself is associated with various adverse reactions like severe hypersensitivity due to histamine release form mast and basophil cells and severe hypotension.18 Also, this PS 80 based formulation leads to non-selective drug distribution and hence gives way to the various toxicities, the most clinically relevant being peripheral neurotoxicities and febrile neutropenia. To address these issues, we report the synthesis of a chondroitin sulphate A – polyethylene glycol conjugate and its subsequent application in the fabrication of docetaxel nanocrystals. The synthesized conjugate mPEG-CSA will offer triple advantage of stabilization, pegylation and CD44 receptor targeting. The developed docetaxel nanocrystals (DTX@CSA-NCs) were suitably characterized and studied in detail in vitro in MDA-MB-231 cells and 4T1 cells. After getting



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convincing results from cell based experiments, and ascertaining that presence of chondroitin has a remarkable effect, the formulation DTX@CSA-NCs was studied in vivo for assessing pharmacokinetics, toxicity, and tumor regression behavior in preclinical animal models. 2. Experimental Section Materials. The drug docetaxel (DTX) was received as a gift sample from Sun Pharmaceutical Advanced Research

Centre,

Baroda.

Chondroitin

dimethylaminopropyl)carbodiimide

(EDC

sulphate HCl)

A

(Himedia),

(TCI

Chemicals),

1-Ethyl-3-(3-

N-Hydroxysuccinimide

(NHS)

(Himedia), Triethylamine (Loba chemie), amine terminated poly(ethylene glycol) (mPEG-NH2) (alfa aesar), Dulbecco’s Modified Eagles Medium (DMEM), antibiotic/antimycotic solution, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), propidium iodide, ribonuclease A, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT), 10X trypsin were purchased from Sigma-Aldrich ( MO, USA). Apoptosis detection kit was purchased from BD Biosciences. Heat inactivated fetal bovine serum (FBS) was procured from Hi-media (Mumbai, India). HPLC grade acetonitrile and methanol were supplied from Merck, India. Methods. Synthesis of the Chondroitin Sulphate- Polyethylene Glycol Conjugate. Chondroitin sulphate A-polyethylene glycol (mPEG-CSA) conjugate was synthesized using carbodiimide chemistry. Briefly, chondroitin sulphate A (CSA) (100 mg), 1-ethyl-3-[36 ACS Paragon Plus Environment

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dimethylaminopropyl] carbodiimide hydrochloride (15.3mg) and N-hydroxy succinamide (9.2 mg) were dissolved in water (5 mL) containing triethylamine (100 µL) under stirring in nitrogen atmosphere at room temperature for activation of –COOH functional group of CSA. After 30 min, mPEG-NH2 (40 mg) dissolved in 3 mL water was added drop wise into the CSA solution. The mixture was further stirred for 48 h, dialyzed against triple distilled water and water/methanol (75/25) mixture for 1 d each followed by lyophilization. The obtained white color product was stored at -20 °C until further use. Fabrication of Docetaxel Nanocrystals. The DTX@CSA-NCs were prepared using high pressure homogenizer (HPH) coupled with heat exchanger. Briefly, DTX (2 mg mL-1) was suspended in the aqueous solution of synthesized conjugate (mPEG-CSA). The suspension was subjected to premilling using high shear homogenizer (12000 rpm for 10 min) and was subsequently processed in HPH at variable pressure and number of cycles to obtain a uniform nanosuspension. Trehalose at a concentration of 5% w/w was used as a cryoprotectant prior to lyophilization of the prepared nanosuspension. The parameters like drug: stabilizer ratio, homogenization pressure and number of cycles were optimized via a series of experiments to obtain a formulation with desired physico-chemical characteristics and high stability. The DTX@PEG-NCs and DTX-NCs were prepared following the same protocol replacing mPEG-CSA with 0.5% PEG and 0.01% PVP and lyophilized for further experimentation. The FITC tagged nanocrystals were prepared by coprecipitating DTX with FITC solution followed by washing and processing in high pressure homogenizer. (Refer supporting information for detailed procedures)



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Particle Size, Zeta Potential and Surface Morphology. The particle size, polydispersity index (PDI) and zeta potential of the prepared batches were determined using Zetasizer 2000 (Nano ZS, Malvern instrument, UK) employing dynamic light scattering principle. All the measurements were done in triplicate at the temperature of 25 ± 1 °C after appropriately diluting the sample. The surface morphology of the prepared DTX@CSANCs was studied using scanning electron microscope (Quanta FEG 450, FEI, The Netherlands) and atomic force microscope (APE Research, Italy). For observation via AFM, a drop of sample was spread on a glass cover slip and air dried. It was then analyzed using non-contact mode. Drug Content. The drug content in the formulated nanocrystals was determined by dissolving 5 mg of lyophilized DTX-NCs, DTX@PEG-NCs or DTX@CSA-NCs into 1 mL of acetonitrile. It was then vortexed for 15 min and centrifuged at 5000 rpm for 10 min. The supernatants were then analyzed after appropriate dilutions using the validated reversed phase HPLC method. The drug content was calculated as: Percent drug content = (Weight of DTX in Nanocrystals/ Total Weight of Nanocrystals) * 100 Measurement of Fix Aqueous Layer Thickness (FALT). The fixed aqueous layer thickness (FALT) of the nanocrystals was estimated by the method reported by Sadzuka et al based on the zeta potential of the nanocrystals in an ionic solution of different concentrations. Briefly, the DTX@CSA-NCs were obtained by ultracentrifugation and washed twice with phosphate buffer saline pH 7.4. The nanocrystals were then resuspended in 8 ACS Paragon Plus Environment

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the sodium chloride solution of different concentration (0mM, 10mM, 20mM, 50mM, 100mM) and the zeta potential was measured using the smoluchowski equation. The FALT was calculated using the correlation between the absolute value, that is magnitude only without regard to sign, of zeta potential (ζ) and Debye-Huckel-parameter (κ), ln ζ = ln A – κL, where A is a constant, L is thickness of ﬁxed aqueous layer in nanometer and κ = √C/0.3, C deno]ng the molarity of univalent salts. 19 In Vitro Dissolution Studies. To assess the drug dissolution kinetics, dialysis bag diffusion method was adopted. Different formulations viz. Taxotere®, DTX coarse suspension and DTX@CSA-NCs equivalent to 2.5 mg of DTX were filled in a dialysis bag (molecular weight cut off 12 KDa) and suspended into the 250 mL dissolution medium, phosphate buffer saline, pH 7.4 (PBS) containing 0.1 % tween 80 to avoid the precipitation of the dissolved drug. The rotation was set at 75 rpm and the temperature was controlled at 37± 0.5 °C. At prefixed time intervals 0.2 mL of dissolution media was collected and replaced with equal volume of media at same temperature to maintain the sink condition. The withdrawn samples were analyzed using developed RP-HPLC method. Storage Stability and Protein corona. The lyophilized nanocrystals were stored at two different conditions of temperature i.e.4 ±2 °C and 22±2 °C up to 8 week to assess the storage stability of the optimized DTX@CSA-NCs. Periodically, a small amount of sample was redispersed in PBS and analyzed for particle size, PDI and zeta potential. In view of the fact that the formulation being developed here is intended for intravenous administration and albumin is the most abundant blood protein, the 9 ACS Paragon Plus Environment


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stability of DTX@CSA-NCs was also assessed in bovine serum albumin (BSA). For this, 10mg of lyophilized DTX@CSA-NCs and non pegylated nanocrystals were incubated with 1mg of BSA for 4 h at 37°C. Afterwards, the suspension was centrifuged at 15,000 rpm to settle down the nanocrystals and the supernatant was analyzed for albumin content employing bicinchoninic based protein assay.20 In Vitro Cell Line Studies Cell Culture Conditions. The MDA-MB-231, MCF-7 and 4T1 cells were procured from ATCC (Rockville, US) and were cultured in DMEM supplemented with 10 % fetal bovine serum and 1% antibiotic/antimycotic solution. The cells were incubated at 37 °C in humidified atmosphere with 5 % carbon dioxide. Time Dependent Cell Uptake Using Confocal Laser Scanning Microscope (CLSM). For this study, MDA-MB-231 cells (1X 102 cells per well) were seeded into poly-l-lysine coated cover slips in twenty four well culture plates with 1 mL of DMEM and allowed to adhere to the cover slips overnight. The cells were then incubated with FITC tagged DTX@CSA-NCs, FITC tagged DTX@PEG-NCs and free FITC for 1 h, 2 h and 4 h. At the end of the respective time duration the cells were washed thrice with PBS to remove surface adhered FITC, fixed with formaldehyde, stained with DAPI and analyzed by CLSM. The images were processed using ImageJ software. Using Flow Cytometer. 10 ACS Paragon Plus Environment

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For analysis using flow cytometer, the MDA-MB-231 and 4T1 cells were plated in six well culture plate (5X 105 cells per well) and incubated for 24 h. Afterwards, the cells were treated with FITC tagged DTX@CSA-NCs, FITC tagged DTX@PEG-NCs and free FITC for 1 h, 2 h and 4 h for MDA-MB-231 and for 1h and 4h for 4T1 in dark. Subsequently, the cell were washed with PBS, trypsinized, resuspended in 500 µL of PBS and analyzed by flow cytometer taking cells without treatment as negative control (BD Biosciences, FACS Calibur, Germany). CD44 Receptor Blocking Study To ascertain that the uptake of DTX@CSA-NCs takes place via CD44 receptors, a receptor blocking study was performed in CD44 positive MDA-MB-231 cells and MCF-7 cells with low expression.21 For this, cells were seeded in six well plates and incubated for 24 h to facilitate cell adhesion. After this, the cells were washed with PBS pH 7.4 and incubated with serum free media with or without 10mg/mL low molecular weight hyaluronic acid (LMW HA, 7.5 KDa) for 1 h at 37 °C in humidified atmosphere. The cell were then washed again and treated with fresh media containing FITC tagged DTX@CSA-NCs and incubated for 2 h. After 2 h the cells were collected by trypsinization, washed to remove any surface adhered particles and dye, resuspended in 500 µL of PBS and analyzed by flow cytometer. Cytotoxicity. The MDA-MB-231 and 4T1 cells at a density of 1 X 104 per well were seeded in the 96 well plate and allowed to attach to the plate surface 24 h prior to the treatment. After 24 h the cells were washed thrice with PBS and treated with different formulations viz. Taxotere® and DTX@CSANCs at varying concentrations. After incubation for 24 and 48 h, the plates were washed with 11 ACS Paragon Plus Environment


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PBS and 10 µL MTT reagent (5mg mL-1) was added to each well followed by further incubation for 4 h at 37 °C. Afterwards, 100 µL of dimethylsulfoxide (DMSO) was added to each well to dissolve the purple formazan crystals and optical density was recorded at 490 nm. The IC50 values were calculated by non-linear regression using graphpad prism 6 (GraphPad Software, Inc, USA). Cell Cycle Arrest. The MDA-MB-231 cells at the density of 1X106 cells were seeded on a 6 well culture plate and incubated overnight to facilitate complete attachment of cells to the culture plate surface. After that it was washed with PBS thrice and treated with Taxotere®, DTX@PEG-NCs and DTX@CSANCs at an equivalent concentration of 0.5 µM. After the requisite incubation period (24 h and 48 h) the cells were again washed with PBS and fixed for 30 min using 70 % pre-cooled ethanol. After fixation, the cells were treated with ribonuclease A (50 µL, 100 µg mL-1) stained with propidium iodide (10 µL, 50 µg mL-1) and analyzed using flow cytometer. Apoptosis. The dual staining, annexin V-FITC dye based method was used to investigate the mode and extent of apoptosis in MDA-MB-231 cells. The cells were seeded in 6 well culture plate and allowed to adhere to the surface overnight. Subsequently, the cells were treated with Taxotere®, DTX@PEG-NCs and DTX@CSA-NCs at a dose of 0.5 µM for 24 h and 48 h. Afterwards, the cells were collected and stained with annexin V-FITC as per manufacturer’s protocol. The cells were finally analyzed by flow cytometer within 30 min of staining.


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Mitochondrial Membrane Potential. The mitochondrial membrane potential (MMP) was measured by employing JC-1 dye to have an insight into the pathway leading to apoptosis. For this purpose, MDA-MB-231 cells were plated in 6 well culture plate and incubated overnight at predefined conditions of temperature and CO2. Afterwards, the media was replaced with fresh media incorporating different formulations of DTX at a dose equivalent of 0.5 µM and the cells were again incubated for 24 h. Following this, the cells were collected, washed and resuspended in 500 µL JC-1 solution (15µg mL-1 in PBS) and analyzed via flow cytometer. Lysosomal Membrane Integrity. To further ascertain the potential of DTX@CSA-NCs to affect caspase independent pathways of cell death, a lysosomal membrane integrity test was carried out using lysosomotropic and weak base acridine orange (AO). The MDA-MB-231 cells were treated with different formulations for 4 h at a dose of 0.5 µM DTX. They were then exposed to 5 µg mL-1 AO for 30 min and analyzed using confocal microscope to detect the leakage of AO into the cytosol. Wound Healing Assay. For this purpose, the MDA-MB-231 cells were seeded in a twelve well culture plate and allowed to reach 85 % confluence. The wound was introduced by scratch based direct manipulation method using a sterile 200 µL pipette tip ( diameter 1 mm, Eppendorf, Germany) applying a constant pressure at an angle of 90°. The cell monolayer was then washed with PBS, treated with different DTX formulations at an equivalent concentration of 0.1 µM, and incubated for 24



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and 48 h. At the end of the experiment the images were acquired with the help of inverted microscope (Nikon Eclipse, Japan). Toxicological Evaluation Histology. At the end of in vivo efficacy studies, the tumor and vital organs viz. liver, lungs, spleen and kidney were isolated from the mice of various treatment groups. The organs were fixed in 10% paraformaldehyde, embedded in the optimal cutting temperature compound (OCT) and sectioned using the cryotome. The collected sections were stained using eosin and hematoxylin solution and observed under the light microscope for histological changes. In Vitro Hemolysis. As the formulation being prepared here is intended for intravenous administration, its effect on the integrity of red blood cells (RBCs) must be studied. For this, fresh blood from SD rats was collected and centrifuged to settle down the RBCs. The RBCs were then washed with PBS twice and resuspended in it to get a 2% v/v. It was then incubated with different concentration of DTX as Taxotere® and DTX@CSA-NCs. Triton-X (1 %) was used as positive control and PBS was used as negative control. The percent hemolysis was calculated by using following formula: Hemolysis (%) = (A sample - A negative control) /(A positive control - A negative control) * 100 Where, A sample, A negative control, and A positive control are absorbances of the sample, negative control and positive control respectively


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Peripheral Neuropathy. Docetaxel induced peripheral neuropathy was studied using behavioral experiments in female balb/c mice according to reported protocol. The animals were injected with saline, Taxotere® or DTX@CSA-NCs every alternate day upto a total of 10 days at a dose equivalent to 10 mg kg-1 of DTX. The change in sensory- motor coordination due to different formulations was assessed employing rotarod test where the latency to falling was measured up to two minutes at a rotating speed of 10 rpm. The test was performed on day 12 and day 26. We also investigated the effects of Taxotere® and DTX@CSA-NCs on heat allodynia in the Eddy’s hot plate. The animals were placed on an Eddy’s hot plate maintained at a temperature of 60 °C and the paw withdrawal responses were counted upto 40 s to avoid any experimentally induced injury to the animals. In Vivo Pharmacokinetics. The in vivo pharmacokinetic study was performed in healthy female SD rats of weight 200 – 250 g. The animals were acclimatized and divided into four groups containing nine animals each. Taxotere®, DTX-NCs, DTX@PEG-NCs and DTX@CSA-NCs at an equivalent dose of 10 mg kg-1 were administered intravenously via tail vein injection to the grouped animals. At predetermined time points( 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 10 h, 12 h) the blood (250 µL) was collected into heparinized microcentrifuge tubes via retro-orbital plexus, centrifuged at 2000 X g and separated plasma was collected. A protein precipitation method using acetonitrile was employed to extract the drug from the plasma. The samples were



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analyzed using reversed phase HPLC and various pharmacokinetic parameters were calculated with the help of WinNonlin software (Pharsight Co., Mountain View, CA, USA). In Vivo Efficacy Studies. To study the effect of the developed formulation in-vivo, 4T1 breast tumors were developed in female balb/c mice. Briefly, 4T1 murine mammary carcinoma cells at the cell density of 1*106 per animal were injected into the lower right abdominal quadrant of mice weighing 18-20 g. When the tumors reached a volume of 100-150 mm3, they were divided into four groups containing 5 animals each. Starting from the 15th day of inoculation, the different groups were treated with saline, Taxotere®, DTX@PEG-NCs and DTX@CSA-NCs respectively at a standard dose of 10 mg kg-1 via tail vein every 3 days up to a total of 15 days and six doses. The study was terminated on day 30 of the inoculation and the tumors were carefully harvested from the mice. Tumor volume as an indicator of the efficacy of different formulation was determined using following formula: Volume (mm3) = π/6 [length (mm) X width2 (mm)] The tumor burden at the end of the study and the body weight of animals throughout the study was also recorded. 3. Results and Discussion Synthesis of the Chondroitin Sulphate A- Polyethylene Glycol Conjugate. The conjugation of CSA with mPEG-amine was confirmed by 1H NMR spectroscopy. Figure 1 displays the synthetic scheme and NMR spectra of CSA and synthesized conjugate. The


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conjugate, mPEG-CSA, formation was confirmed by the presence of strong signal at 3.6 ppm corresponding to-O-CH2-CH2-O- of mPEG-NH2, in addition to the CSA signals.

Figure 1: Synthetic scheme of conjugation between chondroitin sulfate A and polyethylene glycol via carbodiimide chemistry and the NMR spectra of chondroitin sulfate A and prepared pegylated chondroitin (mPEG-CSA). Fabrication and Characterization of DTX@CSA-NCs. Herein, the DTX nanocrystals were fabricated using the synthesized conjugate mPEG-CSA as the stabilizer by employing microfluidization technique. For producing the formulation with desired quality attributes, the process parameters namely homogenization pressure, number of cycles



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and stabilizer concentration were optimized. The effect of these variables on particle size, PDI and zeta potential was observed. The pressure was gradually increased from 1000 bar to 1800 bar with 10 cycles each at 1000 bar, 1200 bar, 1500 bar and 1800 bar. It is evident from figure 2A that the mean particle size and PDI of the DTX@CSA-NCs decreases in a non-linear fashion with increase in both homogenization pressure and number of cycles. After 10 cycles at 1000 bar the particle size was found to be 856 ± 56 nm which finally decreased to 194 ± 9 nm at the end of 10 cycles at 1800 bar (Figure 2C&D). A similar trend was observed with PDI which decreased from 0.782 ± 0.08 to 0.21 ± 0.01 leading to the formation of uniformly sized nanocrystals. The concentration of the stabilizers to be used for any nanoformulation is a critical aspect which demands for a proper investigation.13 Various concentrations of mPEG-CSA were studied for their effect on particle size, zeta potential and stability of DTX@CSA-NCs. It was conveniently observed that with increase in the mPEG-CSA concentration from 0.1% to 0.5% the particle size gradually decreased from 412 nm to 190 nm, zeta potential changed from -9.4 mV to -23.8 mV and colloidal stability of the particles gradually increased (Figure S2). This trend could be attributed to the higher density of the mPEG-CSA leading to the coverage of the entire surface of the nanocrystals thus leading to decrease in irreversible agglomeration via volume restriction or volume exclusion effect. Moreover, when the local concentration of the colloidal particles increases, the solvent molecules diffuses there due to osmotic effect and lead to decreased free energy and increased particle stability which could be further explained on the basis of Napper’s model.22 The conjugate provided steric stabilization which would be helpful in avoiding the formation of protein corona and enhancing the blood circulation time.23 Excellent yield and DTX content amounting to 98.4 ± 0.9 % and 98.03± 0.5 % were achieved for


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DTX@CSA-NCs. The SEM and AFM image (figure 2B) revealed that the nanocrystals were rod shaped with no visible signs of aggregation. The DTX@PEG-NCs were also characterized for particle size, polydispersity index, zeta potential, and drug content which were found to be 195.6 ± 12.5nm, 0.23 ± 0.02, -20.8 ± 4.3 mV and 99.1 ± 0.3%. ( Figure S3)

Figure 2: A) Effect of pressure and number of cycles on the particle size and polydispersity index of the nanocrystals. B) The scanning electron microscopic and atomic force microscopic image of the optimized DTX@CSA-NCs. C and D) The size distribution and zeta potential curve of the optimized nanocrystals. All results are expressed as mean± SD (n=3)



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Fixed Aqueous Layer Thickness. FALT which is a measure of the thickness of the adsorbed polymer layer onto the surface of the nanoparticles was calculated with the help of measured zeta potential. It was observed that with increase in the NaCl concentration from 0 mmol to 100 mmol, the absolute value of zeta potential gradually decreased from 25.3 mV to 2.1 mV and the FALT around the nanocrystals was calculated to be 2.35 nm as presented in figure 3A. This suggests that mPEG-CSA constructed a ‘complete mushroom’ structure onto the nanocrystalline surface and provide stability via steric hindrance. The FALT generated here is also anticipated to reduce the macrophage mediated clearance of nanocrystals and will prolong the systemic residence time. This in turn will positively affect the in vivo performance of the developed DTX@CSA-NCs. In Vitro Dissolution. The in vitro dissolution study was conducted using dialysis bag method to compare the dissolution rate of DTX in different formulations namely Taxotere®, DTX coarse suspension and DTX@CSA-NCs. At the end of 24 h, 52±1.8 % drug was dissolved from DTX@CSA-NCs and 19.54 ± 0.98 % from the coarse drug suspension whereas the release was complete from Taxotere® within 12 h (Figure 3B). The results manifested significantly higher dissolution rate from the DTX@CSA-NCs as compared to coarse drug suspension which can be explained on the basis of Noyes-whitney and Prandtl equation. According to Noyes-whitney equation that is, dX/dt = DA/hD * (Cs-Ct) where dX/dt is the velocity of dissolution, D is the diffusion coefficient, A is the surface area of particle, hD is the diffusional distance, Cs is the saturation solubility and Ct is the concentration around the particles, the increase in the surface area of particles due to


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nanonization will eventually lead to the increased dissolution velocity.24 25 Further, the Prandtl equation says that, hH = k ( L1/2/V1/2), where hH is hydrodynamic boundary layer thickness, k is a constant, L is length of the surface in the direction of flow and V is relative velocity of the flowing liquid against a flat surface. With size reduction during high pressure homogenization, the curvature of the nanocrystals and the L will also decrease. This, in turn, will reduce the hH, leading to a thinner hydrodynamic layer and an increase in surface specific dissolution rate. 26-28 Furthermore, increase in saturation solubility as per Kelvin equation, low propensity for agglomeration, desirable wettability and good redispersibility contribute to the elevated DTX dissolution from nanocrystals.29-30

Figure 3: A) Zeta potential versus Debye-Huckel plot for DTX@CSA-NCs after dispersing them in various concentration of sodium chloride. B) In vitro DTX release from various formulations in pH 7.4 phosphate buffer saline. Stability Studies Storage Stability. 21 ACS Paragon Plus Environment


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The long term stability of the prepared DTX@CSA-NCs was studied under different conditions of temperature periodically measuring particle size, PDI and zeta potential. The lyophilized nanocrystals showed very good redispersibility in PBS without any aggregations and agglomerations. We found (Figure S4 and S5) that the nanocrystals were fairly stable under both conditions of temperature exhibiting little change in size, PDI and zeta potential over the time. Protein Corona. To achieve long term circulation, it is highly desirable for the designed nano-delivery systems to elicit the intended effect in vivo without being recognized and cleared by the immune system due to the formation of the nano-opsonin complex. The DTX@CSA-NCs developed here showed 11.6% protein adsorption as compared to 28.7% in case of the non-pegylated nanocrystals. The effect may, in part, be due to the lesser number of binding sites available on the top of PEG molecules enshrouding the nanocrystals and presence of the FALT and the mushroom morphology of the mPEG-CSA over the nanocrystals. The negative surface charge may also play a significant role. Moreover, a small number of protein molecules which may penetrate the PEG layer will reside inside it, hidden from the particle interface leading to direct interaction between the particles and cells.31 Cytotoxicity. The cytotoxic potential of the developed DTX@CSA-NCs was assessed in CD44 expressing MDAMB-231 human breast cancer cell line and 4T1 murine breast cancer cells after exposing them to various concentration of DTX in different formulation for 24 and 48 h (Table S1 & S2). With 22 ACS Paragon Plus Environment

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MDA-MB-231 cells, at the end of 24 h, the IC50 values were found to be 15.3, 6.2 and 3.6 µg mL1

for Taxotere®, DTX@PEG-NCs and DTX@CSA-NCs. The IC50 values further declined to 10.3, 3.8

and 1.2 µg mL-1 after 48 h of incubation. The results show that targeted nanocrystals achieved 4.3 and 8.6 fold lower IC50 after 24 and 48 h than clinically used formulation. Similarly, for 4T1 cells, the IC50 values were 4.6 and 6.8 fold lower after 24 and 48 h with DTX@CSA-NCs than the Taxotere®. The results are in corroboration with previous reports where higher toxicity with nanoparticles was observed in comparison to the free drug.32 It is worth attributing this enhanced killing to the significantly higher, CD44 receptor mediated uptake of the targeted nanocrystals which could bring higher DTX concentrations inside the cells. The sustained release thereafter, maintained the cytotoxic concentration for a prolonged duration which further lowered the IC50. Time Dependent CD44 Mediated Cell Uptake. The cellular uptake is an important phenomenon in deciding the course of therapeutic efficacy of the developed formulation. Here, the cell uptake was studied in the CD44 expressing MDAMB-231 and 4T1 cells in a time dependent manner by exposing them to FITC solution, DTX@PEG-NCs and DTX@CSA-NCs tagged with FITC (Figure 4 A, B & C). The fluorescence was intensified with passage of time in all treatment groups with the signals being strongest for DTX@CSA-NCs treated group and weakest for the FITC solution. The quantitative flow cytometry results also led to the similar observation with 11%, 25% and 69% positive cells for FITC solution, DTX@PEG-NCs and DTX@CSA-NCs respectively at the end of 2 h and 49%, 65% and 83% after incubation for 4 h with MDA-MB-231 cells and 48.53%, 67.42% and 89.37% after



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4 h incubation with 4T1 cells. The order of uptake in both type of cells, that is, FITC solution < DTX@PEG-NCs < DTX@CSA-NCs confirms the implicit property of the nanoparticulate systems to improve the cellular trafficking of drugs across membranes. Further, the differential fluorescence intensity, indicative of enhanced accumulation of FITC tagged DTX@CSA-NCs in the cytoplasm, could be attributed to the surface coating with chondroitin sulfate A, which acted as a targeting motif complementary to CD44 surface receptor. To further confirm the CD44 receptor mediated uptake we performed receptor blocking studies in MDA-MB-231 and MCF-7 cells in the presence of low molecular weight hyaluronic acid. It was observed that the uptake of DTX@CSA-NCs was higher in the MDA-MB-231 cells as compared to the MCF-7 cells (Figure 4D). The pretreatment with HA did not affect the cellular uptake in MCF-7 cells which could be assigned to the fact that the nanocrystals may have some alternative endocytosis pathway for cellular uptake in MCF-7 cells. However, the cell uptake after HA pretreatment was significantly reduced in the MDA-MB-231 cells clearly indicating that the predominant pathway for the uptake is CD44 mediated endocytosis.


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Figure 4 : A) and B)Dot plot via FACS and fluorescence microscopic images showing FITC positive cells after treating MDA-MB-231 cells with FITC tagged formulations at the end of 1h, 2h and 4 h. C) Dot plot via FACS showing FITC positive cells after treating 4T1 cells with FITC tagged formulations at the end of 1h and 4 h D) Flowcytometry histogram displaying uptake of DTX@CSA-NCs in (D1) MDA-MB-231 and (D2) MCF-7 cells; black: control cells, green: uptake in presence of HA, red: uptake in absence of HA. Cell Cycle Arrest. The taxanes are known to arrest the G2/M phase of cell cycle. Hence we studied the degree of arrest resulting from different formulations after exposing the cells for 24 and 48 h. Whereas 25 ACS Paragon Plus Environment


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the control cells displayed only 1.14 and 2.29 % cells at arrested at G2/M phase, DTX@CSA-NCs showed 45.10% arrest as early as 24 h followed by DTX@PEG-NCs and Taxotere® which arrested 28.33% and 12.57% cell respectively (Figure 5). The fraction increased after 48 h and the G2/M arrest by DTX@CSA-NCs (56.37%) was significantly higher than that induced by DTX@PEG-NCs (33.61%) and Taxotere® (18.21%). The highest degree of cell cycle arrest exhibited by the prepared targeted nanocrystals is, presumably, a result of higher cellular uptake and retention of the formulation leading to higher DTX concentration inside the cells. This in turn will lead to direct binding and stabilization of cellular microtubules and downregulation of free tubulin levels leading to insufficiency of material for mitotic spindle formation followed by cell death. 33

Figure 5 : Histograms showing percent of cells at various stages of cell cycle after treating with different DTX formulations for 24 h and 48 h. 26 ACS Paragon Plus Environment

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Apoptosis. Since apoptosis is one the major mechanism leading to cancer cell death, the assessment of apoptotic potential of different formulations will be of paramount importance. We employed a well reported Annexin V- FITC based assay to quantitatively determine the degree of early and late apoptosis. The results state that the DTX@CSA-NCs induced highest apoptosis in the MDAMB-231 cells after both 24h and 48h amounting to 20.10% early apoptosis and 73.69% late apoptosis (Figure 6A). This higher degree of apoptosis is attributed to the cathepsin mediated mitochondrial pathway of cell death which was most expressed in DTX@CSA-NCs treated cells. 34

Mitochondrial Membrane Potential. MMP is a key indicator of cell health and is central to the apoptotic pathway in cancerous and non-cancerous cells.35 To assess whether or not the higher degree of apoptosis obtained with DTX@CSA-NCs was related with a change in MMP we applied JC-1 dye. Since JC-1 follows Nernstian distribution fashion, a more polarized mitochondria will accumulate more dye leading to aggregation and emission of red fluorescence.36 Results presented in figure 6B state that the mitochondrial depolarization was maximum in DTX@CSA-NCs treated group (58.74%) followed by DTX@PEG-NCs (26.76%) and Taxotere® (7.70%) at the end of 24 h. A similar trend was observed after 48 h where 87.78%, 56.45% and 16.13% cells lost the membrane potential after treatment with DTX@CSA-NCs, DTX@PEG-NCs and Taxotere® respectively. This collapse of transmembrane potential will open the high conductance mitochondrial permeability transition pores (mTPT) leading to the release of apoptotic factors like cytochrome C. Further, the



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enhanced aerobic glycolysis as per ‘Warburg effect’ and increased ROS generation will contribute towards aggravated cytotoxicity.37-38 This confirms that DTX@CSA-NCs act via mitochondrial dependent pathway leading to depolarization and cell death.

Figure 6: A) The dot plot and bar graph representing the percent of MDA-MB-231 cells undergoing early (lower right quadrant) and late apoptosis (upper right quadrant) after treatment with different DTX formulations. B) Dot plot and bar graph showing mitochondrial depolarization at the end of 24 and 48 hour after treatment with Taxotere, DTX@PEG-NCs and DTX@CSA-NCs. The red dots and green dots correspond to normal and depolarized mitochondria respectively.


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Lysosomal Membrane Integrity (LMI). The integrity of lysosomal membrane and its permeabilization plays an important role in the cell death via caspase independent pathways such as activation and release of cathepsins and destabilization of lysosomes. The LMI was assessed using AO and the control cells in figure 7A showed discrete red-orange fluorescence pertaining to the intact lysosomes. However, the groups treated with DTX@CSA-NCs, DTX@PEG-NCs and Taxotere® exhibited a shift in the fluorescence. The results indicate that the DTX@CSA-NCs treated group significantly permeabilized the lysosomes leading to the leakage of AO to cytosolic fraction producing a green fluorescence. The intensity of yellow fluorescence was lesser in other treatment groups. This lysosomotropic property of DTX@CSA-NCs will cause membrane permeabilization and disruption leading to translocation of cathepsin B and D to the cytosol.39 Enhanced cell apoptosis and death via multiple lysosomal dependent pathways will follow.40 Together with the loss of mitochondrial membrane potential, this lysosomal membrane permeabilization will significantly improve the overall outcome of therapy. Cell Migration Assay. The effect of developed DTX@CSA-NCs on the cell invasion and migration owing to the presence of mPEG-CSA was studied via wound healing or scratch assay in highly metastatic CD44 expressing MDA-MB-231 cell line. It was observed that the cells in non treated control wells migrated very fast and filled the entire available cell free space within 48 h of wound induction. In other formulations treatment group migrational suppression was observed and fewer cells were present in the gap with least number of cells in DTX@CSA-NCs treated group



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(figure 7B). This suggests that the DTX@CSA-NCs significantly targeted and altered the expression of CD44 receptors and in turn inhibited cell migration and retarded tumor metastasis.

Figure 7: A) The confocal microscopic images and associated corrected total cell fluorescence showing the disruption of lysosomal membrane in MDA-MB-231 cells after treatment with different DTX formulations and AO staining. B) The scratch based wound healing assay representing metastasis inhibiting potential of the mPEG-CSA stabilized nanocrystals after 24h and 48h.


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In Vitro Hemolysis. The preclinical evaluation to test the compatibility of the developed formulation towards red blood cells is a primary requirement for intravenous administration. Our results suggest that the DTX@CSA-NCs have lesser hemolytic potential than Taxotere® at all the concentrations tested. At the concentration levels of 10 µg mL-1, 50 µg mL-1 and 100 µg mL-1, DTX@CSA-NCs showed 1%, 2.84%, and 3.5% hemolysis compared to 8.3%, 14.5% and 25.7% with Taxotere® (figure 8B). This establishes the acceptable and superior hemo-compatibility of the developed formulation over Taxotere® which is worth attributing to the anionic nature of the nanocrystals and absence of PS-80 in the formulation. Moreover, the reduced hemolysis will also contribute towards the increased safety of the formulation by minimizing the incidences of hemolysis related hypersensitivity reactions. 41-42 Peripheral neuropathy. The peripheral neuropathies are one of the most frequent and dose limiting adverse effect associated with taxanes especially docetaxel. We tested our formulation DTX@CSA-NCs to gain an insight towards its performance against these neuropathies via some behavioral experiments and found that the developed formulation has better neurological safety (Figure 8 C & D). The rota rod test suggested that the motor coordination was not compromised in DTX@CSA-NCs treated group and the performance was comparable to the control group at both time points. The time on rod was 92s and 86s for control and DTX@CSA-NCs treated group at the end of 12 days and 110s and 102s after 26 days. However, Taxotere® treated group showed decreased motor coordination. The assessment of hallmark of CIPN, allodynia,



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replicated the similar observations showing that the DTX@CSA-NCs treated group was least prone to the development of allodynia whereas the group treated with Taxotere® displayed significantly higher pain responses.

Figure 8: A) The histological sections of different organs after treatment with different DTX formulations for 15 days. B) The percent hemolysis resulting with different formulations after incubating them for four hours with 2% red blood cell suspension. C) The evaluation of motor co-ordination studied using rotarod apparatus on day 12 and day 26. D) Pain response in hot plate test after 12 and 26 days.


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Histology. To further assess the in vivo toxicity potential of the developed formulation, the histological analysis was done after staining the tissue sections with eosin and hematoxylin (Figure 8A). The safety and biocompatibility of DTX@CSA-NCs was established as the normal architecture of the organs such as the glomerular structure of kidney and alveolar sacs of lungs were maintained and no signs of necrosis were observed. Whereas the Taxotere® treated group showed some toxicity and necrosis. Also, the tumor treated with the DTX@CSA-NCs showed disarranged and disrupted morphology as compared to control and Taxotere® treated group as a result of higher drug concentration and tumor suppression. Pharmacokinetics. The effect of different nanocrystals on pharmacokinetic parameters upon intravenous administration was determined in SD rats after injecting various formulations at a dose of 10 mg Kg-1. There was no formulation related adverse reaction or mortality during the period of study. The co-plot of plasma concentration profile and various pharmacokinetic parameters are presented in figure 9 and table 2. It was observed that the peak plasma concentration for all formulations was achieved after 5 min of i.v administration. The Cmax was comparable for DTXNCs (9.04 μg mL-1),DTX@PEG-NCs (10.626 μg mL-1), DTX@CSA-NCs (11 μg mL-1) and Taxotere® (10.23 μg mL-1) owing to the increased intrinsic solubility of the nanocrystals. The elimination rate constant (Kel) for DTX@CSA-NCs (0.002623 ± 0.0004 min-1) was significantly lower (P DTX@PEG-NCs>DTX-NCs >Taxotere®. This trend can be attributed to the long circulation time of particulate carrier system over molecular form in blood. There is an absolute mean difference in terms of t1/2 and AUC0-∞ between the treatment groups (DTX@CSA-NCs, DTX@PEG-NCs, and DTX-NCs) indicating the superiority of mPEG-CSA coated nanocrystals over other systems in terms of pharmacokinetics .These results suggest that the DTX@CSA-NCs have a better and favorable pharmacokinetic profile than the clinically used solution formulation.

Figure 9: Plasma DTX concentration versus time profile curve of Taxotere®, DTX-NCs, DTS@PEGNCs and DTX@CSA-NCs. Formulations were injected through intravenous route in rats at a dose equivalent to 10 mg kg-1 of DTX.


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Table 1: Pharmacokinetic parameters obtained after intravenous administration of various DTX formulations at a dose equivalent to 10 mg Kg-1. DTX@CSA-NCs

Parameter

Unit

Taxotere®

DTX-NCs

DTX@PEG-NCs

K Elimination

min-1 min

0.00433 ±0.0015 171.36 ± 49.83

0.00324 ± 0.0009

T1/2

0.005533 ± 0.0024 140.7666 ± 55.96

Tmax

min

5

5

5

5

AUC0-∞

μg/mL*min

806.9026 ± 235.02

945.1 ± 202.57

1293.01 ± 143

1762.955 ± 275.316

226.89 ± 71.74

0.002623 ± 0.0004 267.8474 ± 37.24

In Vivo Tumor Regression. The in vivo tumor regression potential was evaluated in Balb/c mice bearing 4T1 induced tumor. The animal groups received saline, Taxotere®, DTX@PEG-NCs and DTX@CSA-NCs and the tumor volume and the body weight of the animals was regularly monitored. The tumor volume and body weight over the time, morphology of the excised tumors, along with tumor burden and volume at the end of the study is presented in figure 10(A-E). It was observed that all the formulations, Taxotere®, DTX@PEG-NCs and DTX@CSA-NCs led to tumor growth inhibition as compared to the saline administered control group where the tumor growth was rapid. The group treated with DTX@CSA-NCs displayed 19.7, 6 and 4.6 fold tumor growth suppression when compared to control and Taxotere® and DTX@PEG-NCs treated group. Moreover, 20 fold, 5.2 fold and 3.6 fold lower tumor burden was observed in DTX@CSA-NCs treated group as compared to saline treated negative control group, Taxotere® and DTX@PEGNCs respectively. The in vivo findings are in accordance with the in vitro results where the 35 ACS Paragon Plus Environment


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highest efficacy of the DTX@CSA-NCs was established owing to the receptor mediated enhanced cellular uptake and subsequent cytotoxicity. This enhanced internalization is expected to have favored the in vivo performance as well where the volume of the DTX@CSANCs treated group turned out to be the smallest.

Figure 10: A) The mean tumor growth curves during the study, B) The body weight of the animals for the duration of the treatment (C) & (D) The tumor burden and mean tumor volume at the end of the study showing least burden in DTX@CSA-NCs treated group. E) The representative tumor shape and morphology at the end of the study. The values represent n=5 ±SD.


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4. Conclusion Herein, we synthesized a novel chondroitin sulfate A- polyethylene glycol conjugate and successfully applied it as an effective stabilizer for the fabrication of docetaxel nanocrystals. The nanocrystals so developed, DTX@CSA-NCs, displayed acceptable quality attributes in term of particle size, polydispersity index, zeta potential and in vitro drug dissolution. The DTX@CSANCs outperformed non-targeted nanocrystals and clinically used formulation in cell line based assays as the results of enhanced cellular uptake via receptor mediated endocytosis. On probing further, it was evident that the nanocrystals acted via mitochondrial (disruption of membrane potential) and lysosomotropic (disruption of membrane integrity) pathway leading to higher cellular toxicity. The results obtained with in vitro experiments corroborated well with in vivo results where the DTX@CSA-NCs showed higher plasma concentration and higher tumor regression in 4T1 induced mice tumor model. Hence, it could be conveniently concluded that the developed docetaxel nanocrystalline formulation could be a possible approach for better chemotherapy. ASSOCIATED CONTENT Supporting Information Preparation of FITC tagged nanocrystals; Preparation of non-pegylated nanocrystals; Effect of surfactant concentration on particle size and zeta potential of DTX@CSA-NCs; Characterization of DTX@PEG-NCs; Stability studies; Cytotoxicity Studies



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Acknowledgements GP and NM are thankful to CSIR, New Delhi for providing senior research fellowships. We would like to thank Dr. Saman Habib for providing CLSM facility, Mr. A L Vishwakarma for assistance in FACS experiments. Dr. P N Saxena, CSIR-IITR is thankfully acknowledged for helping with SEM imaging. Conflict of Interest None declared. References (1) Lee, J. Y.; Chung, S. J.; Cho, H. J.; Kim, D. D. Phenylboronic Acid-Decorated Chondroitin Sulfate A-Based Theranostic Nanoparticles for Enhanced Tumor Targeting and Penetration. Advanced Functional Materials 2015, 25 (24), 3705-3717. (2) Bae, Y. H.; Park, K. Targeted drug delivery to tumors: myths, reality and possibility. Journal of Controlled Release 2011, 153 (3), 198. (3) Chen, W.; Liu, Y.; Liang, X.; Huang, Y.; Li, Q. Chondroitin sulfate-functionalized polyamidoamine as a tumor-targeted carrier for miR-34a delivery. Acta Biomaterialia 2017. (4) Zhang, X.; Yao, M.; Chen, M.; Li, L.; Dong, C.; Hou, Y.; Zhao, H.; Jia, B.; Wang, F. Hyaluronic AcidCoated Silver Nanoparticles As a Nanoplatform for in Vivo Imaging Applications. ACS applied materials & interfaces 2016, 8 (39), 25650-25653. (5) Chen, H.; Tham, H. P.; Ang, C. Y.; Qu, Q.; Zhao, L.; Xing, P.; Bai, L.; Tan, S. Y.; Zhao, Y. Responsive Prodrug Self-Assembled Vesicles for Targeted Chemotherapy in Combination with Intracellular Imaging. ACS applied materials & interfaces 2016, 8 (37), 24319-24324. (6) Xu, H.; Tian, Y.; Yuan, X.; Wu, H.; Liu, Q.; Pestell, R. G.; Wu, K. The role of CD44 in epithelial– mesenchymal transition and cancer development. OncoTargets and therapy 2015, 8, 3783. (7) Basakran, N. S. CD44 as a potential diagnostic tumor marker. Saudi medical journal 2015, 36 (3), 273279. (8) Huang, W.-Y.; Lin, J.-N.; Hsieh, J.-T.; Chou, S.-C.; Lai, C.-H.; Yun, E.-J.; Lo, U.-G.; Pong, R.-C.; Lin, J.-H.; Lin, Y.-H. Nanoparticle targeting CD44-positive cancer cells for site-specific drug delivery in prostate cancer therapy. ACS applied materials & interfaces 2016, 8 (45), 30722-30734. (9) Lee, J.-Y.; Park, J.-H.; Lee, J.-J.; Lee, S. Y.; Chung, S.-J.; Cho, H.-J.; Kim, D.-D. Polyethylene glycolconjugated chondroitin sulfate A derivative nanoparticles for tumor-targeted delivery of anticancer drugs. Carbohydrate polymers 2016, 151, 68-77. (10) Varghese, O. P.; Liu, J.; Sundaram, K.; Hilborn, J.; Oommen, O. P. Chondroitin sulfate derived theranostic nanoparticles for targeted drug delivery. Biomaterials science 2016, 4 (9), 1310-1313.


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Table of Contents (TOC)

PEGchondroitin coated nanocrystals

CD44 receptor

Depolarized Mitochondria

Lysosome Apoptosis

Nucleus

Release of Cathepsins


Multifunctional Glycoconjugate Assisted Nanocrystalline Drug Delivery

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