Transferrin receptor targeted PLA-TPGS micelles improved efficacy and safety in docetaxel delivery

Accepted Manuscript Title: Transferrin receptor targeted PLA-TPGS micellesimproved efficacy and safety in docetaxel delivery Author: Rahul Pratap Singh Gunjan Sharma Sonali Poornima Agrawal Bajarangprasad L. Pandey Biplob Koch Madaswamy S. Muthu PII: DOI: Reference:

S0141-8130(15)30177-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.11.081 BIOMAC 5586

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

17-10-2015 26-11-2015 27-11-2015

Please cite this article as: R.P. Singh, G. Sharma, P. Agrawal, B.L. Pandey, B. Koch, M.S. Muthu, Transferrin receptor targeted PLA-TPGS micellesimproved efficacy and safety in docetaxel delivery, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.11.081 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Transferrin receptor targeted PLA-TPGS micelles

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improved efficacy and safety in docetaxel delivery

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Rahul Pratap Singha, Gunjan Sharmac,

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Sonalia, Poornima Agrawala, Bajarangprasad L. Pandeya,

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Biplob Kochc, Madaswamy S. Muthua, b*

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Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, U.P., India

b

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Department of Pharmaceutics, Indian Institute of Technology, Banaras Hindu University, Varanasi – 221005, India

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Department of Zoology, Faculty of Science, Banaras Hindu University, Varanasi 221005, U.P., India

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_______________________________________ *

Corresponding author: Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi – 221005, India Tel.: +91 9235195928; Fax: +91 542 2367568; E-mail: [email protected] (Madaswamy S. Muthu).

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 1

Page 1 of 40

ABSTRACT

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The aim of this work was to develop targeted polymeric micelles of poly-lactic acid-D-

3

α-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS), which are assembled along with

4

D-alpha-tocopheryl polyethylene glycol 1000 succinate-transferrin conjugate (TPGS-Tf), and

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loaded docetaxel (DTX) as a model drug for enhanced treatment of lung cancer in comparison to

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non-targeted and DTX injection (DocelTM). A549 human lung cancer cells were employed as an

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in-vitro model to access cytotoxicity study of the DTX loaded polymeric micelles. The safety of

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DTX formulations were studied by the measurement of alkaline phosphatase (ALP), lactate

9

dehydrogenase (LDH) and total protein levels in bronchoalveolar lavage (BAL) fluid of rats after

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the treatments. The IC50 values demonstrated that the non-targeted and transferrin receptor

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targeted polymeric micelles could be 7 and 70 folds more effective than DocelTM after 24 h

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treatment with the A549 cells. Results suggested that transferrin receptor targeted polymeric

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micelles have showed better efficacy and safety than the non-targeted polymeric micelles and

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DocelTM.

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Key words: Cytotoxicity, Lung cancer, Nanomedicine, Nanotechnology, Polymeric micelles,

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Safety, Transferrin conjugation

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1. Introduction Lung cancer is one of the top most deaths causing disease in the world in both sexes (men Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 2

Page 2 of 40

and women). Based on lethality, the lung cancer is further categorized in two major classes

2

including small cell lung cancer (13%) or non-small cell lung cancer (83%) for the purposes of

3

treatment. The 1 and 5 year relative survival rates for lung cancer are 44% and 17%, respectively.

4

The lung cancer treatment includes surgery, radiation therapy, chemotherapy, and/or targeted

5

therapies. The lung cancer patients are usually treated by chemotherapy and targeted drugs [1].

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Presently, various receptor targeted drug delivery systems have showed the possibilities to provide

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effective treatments to cancer cells which may minimize the adverse effects of drugs to the healthy

8

cells [2].

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The nanomedicine is the application of nanotechnology to medicine, which is highly

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capable to develop nanoparticles drug delivery system [3]. These nano drug deliveries are enable

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to target cancer cells in highly effective manner by enhanced permeability and retention (EPR)

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effects, which is an important feature to make cancer targeting drug delivery system [4-10]. The

13

several

14

(D,L-lactide-co-glycolide) (PLGA) nanoparticles, carbon nanotubes and polymeric micelles

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[11-22]. Among these, polymeric micelles are drug delivery systems, which are widely applied in

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the lipophilic or poorly soluble drugs [17,23-25].

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systems

have

been

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delivery

developed

such

as

liposomes,

poly

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drug

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The polymeric micelles are formed through the multimolecular assembly of block

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copolymers as novel core-shell typed nano carriers for drug targeting [26,27]. The polymeric

19

micelles outer corona made up by using the hydrophilic polymer segments, which act as a

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stabilizing interface between the hydrophobic drug and the external medium and maintains the

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aqueous solubility of polymeric micelles [28-30].

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Polymeric micelles are emerging polymeric nano carriers, which act as a highly integrated

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nanoplatform for cancer targeting (passive as well as active targeting), drug delivery and tumor

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 3

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imaging applications [31-33]. It have several extraordinary properties including biocompatibility,

2

high stability in both in-vitro and in-vivo, and also have capacity to effectively solubilize a variety

3

of poorly soluble drugs, changing the release profile of the incorporated pharmaceutical agents,

4

and the ability to accumulate in the target zone based on the EPR effects [34-35]. These polymeric

5

micelles can be used as targeted drug delivery systems in lung cancer treatment [17, 36]. Also, it

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can be conjugated with different biomolecules including targeting ligand, antibody,

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P-glycoprotein (P-gp) inhibitor and diagnostic agents, which are highly beneficial to multiple drug

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resistance (MDR) types of lung cancer treatment [37-39]. Also, polymeric micelles are highly

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effective to increase EPR effect in cancer treatment. The polymeric micelles are frequently using

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in the several cancer treatment including glioma, renal, stomach, lung, and pancreatic cancer at

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phase I and phase II clinical trials. [28, 40, 41].

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Poly-lactic acid (PLA) is a biodegradable and biocompatible polymer, which is widely

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using in the biomedical for making safe and biodegradable nano carriers for cancer treatment. The

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United States Food and Drug Administration (US FDA) has approved their uses in drug delivery

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systems. The monomer of PLA i.e., L-lactic acid (L-LA), which can be efficiently produced by

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fermentation from renewable resources such as starchy materials and sugars. The PLA degrades to

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its monomer, L-LA, which is a normal metabolite of the human [13]. D-α-tocopheryl polyethylene

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glycol 1000 succinate (TPGS) mostly studied in liposomes and polymeric nanoparticles which

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enhanced cellular uptake, cytotoxicity and showed prolonged circulation time [14,15]. TPGS is

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simply known as vitamin E, which is provides better permeation effects of hydrophilic and

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hydrophobic drug molecules. It is FDA approved water-soluble derivative of natural Vitamin E

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(PEGylated vitamin E). TPGS is a derivative of the natural vitamin E (alpha-tocopherol) and

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polyethylene glycol 1000. It is also non-ionic and highly hydrophilic, is used as solubilizer,

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absorption enhancer and a vehicle for various formulations [13,15,22]. PLA-TPGS is a block

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copolymer of L-LA and TPGS. The PLA-TPGS block-copolymer was prepared by using ring

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opening polymerization method. It has been used in the fabrication of nano drug delivery systems

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because of its safety, stealth effect, targeting feasibility, multifunctional capability,

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biocompatibility, size tunability and ease of preparation of nanoparticles. In recent study, de

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Melo-Diogo et al., has prepared a highly effective drug delivery of triple multiple drugs

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(crizotinib–palbociclib–sildenafil) loaded PLA-TPGS micelles and improved lung cancer

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treatment

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(Crizotinib–Palbociclib) loaded micelles treatment [17]. The polymeric micelles of PLA-TPGS

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can be prepared at the critical micelles concentration (CMC) of 0.016 mg/mL [17]. Further,

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targeting can be achieved via the EPR into the areas with the compromised vasculature and by

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attaching specific targeting ligand molecules to the micelle surface [13, 42-44].

A549

cells

in

comparison

to

single

(Crizotinib)

and

dual

drug

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In this study, docetaxel (DTX) was used as anticancer model drug for the lung cancer

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treatment. DTX (N-debenzoyl-N-tertbutoxycarbonyl-10-deacetyl-paclitaxel) is a semisynthetic

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derivative of the taxoid family of antineoplastic agents. It is an analog of paclitaxel, which is

16

extracted from the needles of the European yew tree (Taxus baccata L). DTX has been found to be

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more effective than paclitaxel against breast, ovarian, lung, brain and neck cancers [15, 16]. To

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overcome the poor solubility of DTX and to reduce the systemic toxicity, conventional and various

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novel formulations have been developed [20, 22, 45]. However, there is a problem that needs to be

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solved to meet the requirements for its clinical use i.e., targeted delivery of DTX to cancer cells.

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Transferrin receptor is a cell membrane-associated glycoprotein on the surface of the cells. It

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plays an important role in iron homeostasis and the regulation of cell growth in cells. The

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transferrin receptor expression on cancer cells can be up to 100-fold higher than the average

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 5

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expression in normal cells owing to their rapid proliferation rate and iron demand, thereby

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transferrin has been explored as a targeting ligand for nanocarriers to deliver therapeutic and

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diagnostic agent into cancer cells [46,47]. This approach shows the advantage of continuous

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recycling of the transferrin receptor from the surface to the endosomal compartment to make it

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efficient route for the internalization of nanoplatforms [20]. In one study, it was reported that

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among the primary lung cancer cases, 19% of adenocarcinomas showed positive results for

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transferrin receptor overexpression which showed the possibility of transferrin receptor targeted

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delivery of micelles for lung cancer therapy [48].

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In this study, PLA-TPGS (block copolymer of L-LA and TPGS) was used to make polymeric

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micelles. DTX was loaded into polymeric micelles by using solvent displacement method.

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Transferrin was conjugated to activated TPGS-COOH, which serve as targeting agent to transport

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DTX into the human lung cancer cells (A549). The targeting effects of transferrin conjugated

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polymeric micelles (targeted) were compared closely with non-targeted polymeric micelles and

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clinical DocelTM (DTX injection). In-vitro cytotoxicity of A549 cells was assessed and the IC50

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value at 24 h was assessed to evaluate the therapeutic effect of the DTX loaded PLA-TPGS

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polymeric micelles with or without targeting function. Further, PLA-TPGS micelles were tested

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in-vivo on rats model for safety applications i.e., alkaline phosphatase (ALP), lactate

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dehydrogenase (LDH), and total protein levels in bronchoalveolar lavage (BAL) fluid of lung

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tissue after intravenous (i.v.) administrations.

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2. Materials and methods

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2.1. Materials

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Docetaxel (DTX) of purity 99.56% was obtained from Neon Laboratories Ltd, Mumbai, India

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as gift sample. D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) was obtained from

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Antares Health Products., St. Charles, U.S.A. as gift sample. Dialysis membrane (Spectra/Por7®)

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of 1 KDa molecular weight cut off was purchased from Spectrum Laboratories Inc., Rancho

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Dominguez, CA, U.S.A. Clinical formulation DocelTM was purchased from RPG Life Sciences

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Limited, Mumbai, India. L-Lactide (L-LA) monomer (3,6-Dimethyl-1,4-dioxane-2,5-dione),

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stannous octoate (Sn(Oct)2), transferrin (Molecular weight of 80 kDa), ethanol and phosphate

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buffer saline (PBS) were purchased from Sigma–Aldrich, St. Louis, MO, USA. Anhydrous

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toluene, dichloromethane and methanol were purchased from Loba Chemie Pvt. Ltd, New Delhi,

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India. Human lung cancer cell lines (A549) were provided by National Centre for Cell Science,

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Pune, India. Dulbecco's Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS),

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streptomycin, penicillin, L-glutamine and trypsin-EDTA were purchased from Genetix Biotech

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Asia Pvt. Ltd., Mumbai, India. T-25 culture flask and 96-well culture plates purchased from

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Tarsons

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5-diphenyltetrazoliumbromide (MTT) were purchased from Himedia Laboratories, Mumbai,

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India. All other chemicals were of analytical grade.

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2.2. Methods

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2.2.1. Synthesis of PLA-TPGS copolymer

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Ltd.,

Kolkata,

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Pvt.

India.

3-(4,

5-dimethylthiazolyl-2-yl)-2,

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Products

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The PLA-TPGS copolymer was synthesized by ring opening polymerization (ROP) of

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L-LA with TPGS in the presence of stannous octoate [Sn(Oct)2] as catalyst. Briefly, L-LA and

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TPGS (1:1 ratio) were added to a round bottom flask (RBF). The system was purged with nitrogen

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and anhydrous toluene (25 mL per gram of L-LA) was added. Subsequently Sn(Oct)2 (0.5% w/v)

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was added and the ROP reaction was carried out at 120 0C for 4 h, under nitrogen (N2) atmosphere.

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At the end of polymerization, the toluene was evaporated by using rotary evaporator (Buchi R-210

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 7

Page 7 of 40

Advanced, Switzerland) at 37 0C and the resulting product was dissolved in dichloromethane

2

(DCM) and precipitated in cold methanol. The recovered precipitate was dialyzed for 2 days

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against acetone and 3 days against distilled water. Finally, the diblock copolymer was lyophilized

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and a white powder was obtained [11,17,41,47,49]. The schematic diagram of PLA-TPGS

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copolymer synthesis resents in Fig. 1A.

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2.2.2. Synthesis of TPGS-COOH (activation of TPGS) and its conjugate

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For the synthesis of TPGS and transferrin conjugate, TPGS was first activated (i.e.,

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TPGS-COOH) by succinic anhydride through ring-opening reaction in the presence of DMAP

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[20]. In brief, TPGS (0.77 g, 0.5 mM), succinic anhydride (0.10 g, 1 mM) and DMAP (0.12 g, 1

10

mM) were mixed and heated at 100 0C under nitrogen atmosphere for 24 h. The mixture was

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cooled to room temperature, dissolved in 5 ml of cold dichloromethane, filtered to remove

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excessive succinic anhydride and then precipitated in 100 ml of diethyl ether at -10 0C overnight.

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The white TPGS-COOH precipitate was filtered and dried in vacuum [19,20,22]. The schematic

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diagram of synthesis of TPGS-COOH (activation of TPGS) resents in Fig. 1B (i).

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Further, TPGS-COOH carboxyl group was conjugated to the transferrin by carbodiimide

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chemistry with the help of EDC and NHS in phosphate buffer saline (pH 5.5) [20]. In brief, for

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conjugation of transferrin to TPGS-COOH, 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide

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(EDC) and N-hydroxysuccinimide (NHS) (both are catalyst) were added into TPGS-COOH with a

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molar ratio of 1:5 (TPGS-COOH : EDC or NHS) in phosphate buffer saline (pH 5.5). In brief,

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TPGS-COOH (200 mg), EDC (96 mg) and NHS (74 mg) were mixed in 2 ml of phosphate buffer

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saline (pH 5.5) at 25 0C for 5 h, stored in a refrigerator at 4 0C over 24 h, mixed with 1 ml of 0.2 %

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(w/v) transferrin and stirred at 4 0C over 8 h. The resulted product was then dialyzed using a

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dialyzing membrane (MWCO: 12 kDa) against phosphate buffer saline (pH 5.5) for 48 h in order

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 8

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to remove excess TPGS-COOH, NHS and EDC. The dialyzed product (i.e., TPGS-Tf) was freeze

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dried and used for micelle formation [20,22]. The schematic diagram of synthesis of TPGS-Tf

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resents in Fig. 1B (ii).

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2.2.3. Preparation of PLA-TPGS polymeric micelles using solvent displacement (SD) method

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Briefly, for DTX loaded polymeric micelles formulation, 3 mg DTX and 25 mg

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PLA-TPGS copolymer were dissolved in 5 ml of DCM/methanol (1:1 v/v). After that, the solvent

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was evaporated by using rotary evaporator (Buchi R-210 Advanced, Switzerland) at 37 0C and

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then film was hydrated with 10 ml of 1mM phosphate buffer saline (PBS), pH 7.4. After that the

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mixture was incubated in an orbital water bath shaker at 37 0C for 48 h. The excess

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non-incorporated DTX was separated by filtration (using a 0.22-µm filter) before characterization.

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The targeted polymeric micelles were prepared with the addition of 10 mg of TPGS-Tf at 4 0C to

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obtain higher drug encapsulation, drug controlled release and drug targeting effects on cancer cell

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lines (Table 1). For the preparation of placebo polymeric micelles, the formulation process used

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was the same as previously described but without the inclusion of DTX [16,17]. The schematic

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diagram of preparation of non-targeted and targeted polymeric micelles resents in Fig. 1C (i) and

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(ii), respectively.

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2.2.4. Fourier transform infrared (FTIR) spectroscopy

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The FTIR spectroscopy for L-LA, TPGS, PLA-TPGS copolymer, TPGS-COOH and

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TPGS-Tf were performed using compressed KBr pellet method in Perkin Elmer FTIR

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spectrophotometer (Perkin Elmer Spectrum Two, Waltham, Massachusetts, U.S.A). The scanning

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range was 400-4000 cm-1.

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2.2.5. Particle size, polydispersity and zeta potential of polymeric micelles

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Size, polydispersity and zeta potential of the PLA-TPGS (placebo micelles),

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DTX-PLA-TPGS (non-targeted micelles) and DTX-PLA-TPGS-Tf (targeted micelles) were

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measured by PCS using a Zetasizer ver. 7.03, Malvern instruments Ltd, Malvern, UK [16].

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2.2.6. Determination of encapsulation efficiency and drug loading of polymeric micelles The DTX encapsulation efficiency of non-targeted and targeted polymeric micelles were

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determined by UV spectrophotometer (Shimadzu 1800, Tokyo, Japan). Briefly, 2 ml aliquots of

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polymeric micelles were evaporated to dryness by rotary evaporator under reduced pressure at 35

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0

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h at 37 0C to fully leach out the DTX. Then, the solution was centrifuged at 10,000 rpm for 10 min

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at 4 0C, and the supernatant was collected. The supernatant (500 µl) was further diluted to 2 ml of

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methanol. Then light absorption was measured at 229 nm using spectrophotometer. The drug

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encapsulation efficiency was defined as the ratio between the amount of DTX encapsulated in the

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polymeric micelles and that added in the polymeric micelles preparation process. The drug loading

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was calculated as the weight of drug (µg) per one mg of the drug-loaded polymeric micelles

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[20,22]. All samples were done in triplicate.

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2.2.7. In-vitro drug release studies

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The release patterns of DTX from DTX-PLA-TPGS and DTX-PLA-TPGS-Tf were studied

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by using dialysis bag diffusion technique [15,22]. The formulation of a volume equivalent to 300

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µg of DTX were placed in the dialysis bag (cellulose membrane, molecular weight cut off 1 kDa),

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hermetically sealed and immersed into 100 ml of PBS (pH 7.4). The entire system as kept at 37 ±

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0.5 0C with continuous shaking at 100 rpm/min. Then, 5 ml samples were withdrawn from the

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receptor compartment at predetermined time intervals and replaced by fresh medium. The samples

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were filtered through 0.45 mm syringe filter before analysis. The DTX content in the samples was

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determined by using UV spectrophotometer (Shimadzu 1800, Tokyo, Japan). Then drug release

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 10

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profiles were calculated [20, 50].

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2.2.8. Cell culture A549 human lung cancer cell lines were grown in DMEM supplemented with 10% FBS,

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100 units/mL penicillin and 100 µg/mL streptomycin solutions. The cell lines were grown at 37 °C

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in the presence of 5% CO2.

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2.2.9. Cytotoxicity of PLA-TPGS polymeric micelles

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The cytotoxicity of DTX formulations i.e., DTX-PLA-TPGS, DTX-PLA-TPGS-Tf and

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DocelTM (DTX injection) were performed on A549 human lung cancer cell lines by standard MTT

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assay. A549 cell lines were seeded into 96 well culture plates (Tarsons Products Pvt. Ltd.) at the

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density of 1×104 viable cells/well with DMEM and incubated at least overnight to allow cell

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attachment. The spent medium was discarded and the cells were incubated with both

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DTX-PLA-TPGS and DTX-PLA-TPGS-Tf, and their cytotoxicity was assessed in comparison

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with DocelTM at 0.025, 0.25, 2.5 and 25 µg/ml equivalent drug concentrations for 24 h. After 24 h,

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medium was removed and 100 µL medium and 10 µL of 5 mg/mL MTT in PBS, pH 7.4 was added

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to each well of the plate. The plates were further incubated for 2 h at 37 0C in the incubator. Then

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the medium and MTT were removed and 100 µL of DMSO was added to dissolve the MTT

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formazan crystals. The absorbance of samples was measured at 570 nm by microplate reader [23].

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Cytotoxicity was calculated as the percentage of treated cells relative to untreated cells at 570 nm.

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The percent of the cell viability was calculated using the equation:

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Cell viability (%) = Absorbance of treated cells/Absorbance of control cells) X 100 2.2.10. In-vivo safety using bronchoalveolar lavage (BAL) fluids of rats

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The animal caring, handling, husbandry and the protocols were approved by the Institute of

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Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi, India. Both sexes (male and

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 11

Page 11 of 40

female) rats of 150-200 gm (or 4–5 weeks old) were supplied by the Central Animal House of

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IMS, BHU and maintained at animal room of the Department of Pharmacology of IMS, BHU,

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Varanasi, India. The animals were acclimatized at a temperature of 25 ± 2 0C and a relative

4

humidity of 50–60% under natural light/dark conditions and given aseptic full-price nutritional

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pellet feed and sterile water available ad libitum for 4–5 days before experiments. The study

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protocol was follow guideline of Committee for the Purpose of Control and Supervision on

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Experiments on Animals (CPCSEA; Guidelines for Laboratory Animal Facility) Registration No.

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Dean/2015/CAEC/1262 of Banaras Hindu University, Varanasi (U.P.), India.

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The animals were randomly divided into 5 (4 treatment groups + 1 control group) groups

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with approximately 4 rats per group (n=4). One week after the initiation of acclimatization, all rats

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(except control animal) were treated with plain PLA-TPGS (placebo micelles), DTX-PLA-TPGS

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(5 mg/kg of DTX), DTX-PLA-TPGS-Tf (5 mg/kg of DTX) and DocelTM (5 mg/kg of DTX) and

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one control group was treated with normal saline (0.9% NaCl) solution via the i.v. route. Drinking

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water was available ad libitum. During the treatment, the health conditions of rats were monitored

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every day. At the indicated time of interval 7, 15 and 30 days of polymeric micelles and DocelTM

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formulation administrations. The rats were sacrificed with an intraperitoneal (i.p.) injection of

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urethane (1.5 g/kg rats). The thorax was opened and the lung perfused with normal saline solution

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(26.5 ml/kg rats). Further trachea was then cannulated and BAL fluid was performed by injecting

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approximately 4 mL of normal saline solution and thorough rinsing of the lung. The BAL fluid

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was recovered and centrifuged at 400×g at 4 0C for 10 min and then stored at -20 0C until analysis

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[50]. All biochemical assays were performed on BAL fluids such as estimation of ALP (Span

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Diagnostics Ltd., Surat, India), LDH and total protein count (Crest Biosystems, Goa, India) by

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using respective diagnostic kits according to the manufacturer’s instructions. ALP activity is a

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measure of type II alveolar epithelial cell secretory activity, and increased ALP activity in BAL

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fluids is considered to be an indicator of type II cell toxicity. LDH is a cytoplasmic enzyme and is

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used as an indicator of cell injury. Increases in BAL fluid protein concentrations generally were

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consistent with enhanced permeability of vascular proteins into alveolar regions [51, 52].

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2.2.11. Statistical analysis

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Results are given as mean ± standard deviation (S.D). Mean values of particle size,

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encapsulation efficiency, in-vitro and in-vivo data were compared using the Student’s t-test.

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Differences are considered significant at a level of P<0.05.

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3. Results and discussions

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3.1. Preparation of different block-copolymer, TPGS-Tf and polymeric micelles

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Fig. 1A shows the schematic production of PLA-TPGS block-copolymer, that was

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prepared by using ring opening polymerization method in presence of stannous octoate (Sn(oct)2)

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as catalyst in the reaction under nitrogen gas environment at 120 0C for 4 h [17,48]. In the

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polymerization reaction hydroxyl terminus (-OH) of TPGS was used as initiator. Further, this

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amphiphilic copolymer of PLA-TPGS was used to prepared polymeric micelles. Fig. 1B (i) shows

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the activation TPGS in to TPGS-COOH in the presence of succinic anhydride and DMAP at 100

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0

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attached to the activated TPGS (TPGS-COOH) by carbodiimide chemistry with the help of EDC

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and NHS in phosphate buffer saline (pH 5.5) [20]. Fig. 1C (i and ii) clearly shows the preparation

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DTX loaded polymeric micelles. The DTX was loaded using solvent displacement (SD) method.

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C under nitrogen atmosphere for 24 h [22]. Fig. 1B (ii) shows that transferrin was covalently

22 23

Table 1 (approximate position)

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 13

Page 13 of 40

1

Fig. 1 (approximate position)

2 3

3.2. Characterization of L-LA, TPGS, PLA-TPGS, TPGS-COOH and TPGS-Tf by FTIR Fig.2A shows the FTIR spectra of L-LA, TPGS and PLA-TPGS, which is clearly revealed

5

the successful polymerization of L-LA and TPGS in to PLA-TPGS block-copolymer. In the

6

spectra, the C-H stretching is clearly appearing, which is due to methyl group vibrations from

7

L-LA (2925.55 cm-1), TPGS (2870.67 cm-1) and PLA-TPGS (2926.62 cm-1). The carbonyl

8

vibration peak was also present in all the spectra. The carbonyl band shift from TPGS to the

9

synthesized diblock-copolymer (PLA-TPGS) was also visible (TPGS: 1737.75 cm-1; PLA-TPGS:

10

1755.73 cm-1; L-LA: 1766.55 cm-1). Another important peak for -C-C- stretching is aromatic ring

11

was appeared in both TPGS and PLA-TPGS copolymer peak at 1465.06 and 1455.21 cm-1,

12

respectively. The peak at 1382.4 cm-1for -CH2 group of PEG chain was observed in PLA-TPGS

13

copolymer. In TPGS and PLA-TPGS FTIR spectra, the C-O stretch band is also present at peak

14

951.46 and 1252.4 cm-1, respectively. Finally at peak 3425.51 cm-1 band is assigned to the terminal

15

hydroxyl group of the PLA chain.

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Fig.2B shows FTIR spectra of TPGS-COOH and TPGS-Tf. In FTIR spectra of

17

TPGS-COOH, the –C=O stretching was clearly appeared peaks at 1736.47 cm-1, which is clearly

18

indicating a successful synthesis of TPGS-COOH. In transferrin conjugated TPGS, the linkage

19

between -COOH group of TPGS and -NH2 group of transferrin was confirmed by the amide

20

(-CO-NH-) stretching peak at 1634.02 cm-1. In both TPGS and TPGS-COOH, the absorption

21

bands at 3450.04 and 3452.3 cm-1 were due to the terminal hydroxyl group.

22 23

Figure 2 (approximate position)

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 14

Page 14 of 40

1 2

3.3. Particle size, polydispersity and zeta potential of polymeric micelles The mean particle size and polydispersity of PLA-TPGS (placebo micelles),

4

DTX-PLA-TPGS and DTX-PLA-TPGS-Tf were listed in Table 2. PCS measurements were

5

undertaken in multimodal analysis to get a true reflection of particle size distribution. The particle

6

size distribution curves for all the samples were unimodal. The mean sizes of PLA-TPGS,

7

DTX-PLA-TPGS, and DTX-PLA-TPGS-Tf were 84.09 ± 7.3, 135 ± 6.5 and 184 ± 9.92 nm,

8

respectively (Fig.3A to C). The PLA-TPGS polymeric micelles were observed in very small size,

9

which are suitable for therapeutic application in cancer treatment [14,16]. The particle size results

10

indicate that incorporation of DTX and targeting ligand (transferrin) conjugation on the surface of

11

targeted polymeric micelles (DTX-PLA-TPGS-Tf) leads to significant (P < 0.05) increase in the

12

size in comparison to DTX-PLA-TPGS and PLA-TPGS. The size of non-targeted and targeted

13

polymeric micelles were bigger that the placebo polymeric micelles. This results may be due the

14

packaged micelles contained the more hydrophobic drugs in their cores, thus increasing the

15

diameter of the micelles in aggregation. It was reported that polymeric micelles size typically in

16

the range of ˂200 nm are suitable for drug delivery in cancer therapy. The polydispersity of all the

17

batches of polymeric micelles showed quite narrow size distribution, which is nearer to 0.5

18

(Fig.3B). The zeta potential of all the polymeric micelles were found to be negatively charged

19

except for the transferrin conjugated polymeric micelles (Table 2). The negative zeta potential of

20

plain was found to be higher than that of DTX loaded polymeric micelles owing to the better

21

packing of -OH on the surface of polymeric micelles. Also the raise in positive charge on the

22

transferrin conjugated polymeric micelles was due to the presence of amino ends on the surface

23

[20].

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Page 15 of 40

1

3.4. Determination of encapsulation efficiency and drug loading of polymeric micelles Drug encapsulation efficiency was calculated as the percentage ratio between amount of

3

drug loaded in polymeric micelles and amount of the drug added during fabrication of polymeric

4

micelles [28,41,45]. Fig.3D shows the drug encapsulation efficiency of the DTX-PLA-TPGS and

5

DTX-PLA-TPGS-Tf were 71.14 ± 6.7 and 86.63 ± 4.2%, respectively (Table 2). The targeted

6

polymeric micelles (DTX-PLA-TPGS-Tf) showed significant increase (P<0.05) in the drug

7

encapsulation in comparison to non-targeted polymeric micelles (DTX-PLA-TPGS). This may be

8

because of the influence of TPGS-Tf conjugate to surface of polymeric micelles that leads to

9

enhance drug encapsulation efficiency. Drug loading is defined as the weight of drug (µg) per mg

10

of the drug loaded polymeric micelles. The drug loading of the non-targeted polymeric micelles

11

(DTX-PLA-TPGS) was 37.23±3.2 µg/mg. The drug loading of the targeted (DTX-PLA-TPGS-Tf)

12

polymeric micelles was 39.69 ± 2.4 µg/mg (Table 2). As mentioned earlier, addition of TPGS-Tf

13

into the micelle formation might lead to slight increase (P < 0.05) in drug loading and drug

14

encapsulation efficiency for DTX-PLA-TPGS-Tf comparison to DTX-PLA-TPGS.

16 17 18 19

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Table 2 (approximate position)

Figure 3 (approximate position)

3.5. In-vitro drug release studies

20

Fig. 4 shows the accumulated percentage release of DTX from polymeric micelles and

21

DocelTM in the medium of PBS (pH 7.4) under sink condition. The DTX-PLA-TPGS and

22

DTX-PLA-TPGS-Tf showed a sustained release for 72 h without any burst release in comparison

23

to DocelTM. After 72 h of dialysis in PBS (pH 7.4), the percentage of DTX released from the

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 16

Page 16 of 40

preparations were 90.35, 66.72 and 58.23% for the DocelTM, DTX-PLA-TPGS, and

2

DTX-PLA-TPGS-Tf, respectively. The DTX-PLA-TPGS showed sustained drug release effects

3

due to a strong interaction between the DTX and polymeric micelles in comparison to DocelTM.

4

There was a significant (P<0.05) decrease in the in vitro drug release from the

5

DTX-PLA-TPGS-Tf comparison to the DTX-PLA-TPGS and DocelTM. These results may be

6

explained by the influence of transferrin on the micelles surface which was included as TPGS-Tf.

7

The t50% in PBS (pH 7.4) for DocelTM, polymeric micelles of DTX-PLA-TPGS and

8

DTX-PLA-TPGS-Tf containing DTX were about 16, 44 and 58 h, respectively. The DocelTM did

9

not show sustain the drug release. There was a significant (P<0.05) decrease in the t50% of the

10

DTX-PLA-TPGS, and DTX-PLA-TPGS-Tf in comparison with the DocelTM. It may be because of

11

PLA-TPGS structure polymeric micelles, which have followed sustained drug release in PBS up to

12

72 h. Interestingly, the sustained drug release kinetics of DTX-PLA-TPGS-Tf was related to the

13

transferrin conjugate on the micelles [20].

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Figure 4 (approximate position)

3.6. Cytotoxicity of drug formulations

18

In-vitro cytotoxicity in the A549 human lung cancer cells was assessed after 24 h

19

incubation with the DTX formulated in the DTX-PLA-TPGS and DTX-PLA-TPGS-Tf at 37 0C in

20

comparison with that of DocelTM at the equivalent drug concentration. The results are presented in

21

Fig. 5. It is worthy to note that the DTX-PLA-TPGS achieved the higher cytotoxicity compared

22

with the case of DocelTM treatment. This could be due to the effect of enhanced cellular uptake and

23

sustained drug release manner of DTX from the PLA-TPGS polymeric micelles structure.

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 17

Page 17 of 40

Interestingly, DTX-PLA-TPGS-Tf resulted in further raise in cytotoxicity for A549 cells

2

compared with the DTX-PLA-TPGS. It is straightforward to understand that the increase in drug

3

concentration from 0.025 µg/ml to 25 µg/ml resulted in targeted and higher cytotoxicity for

4

DTX-PLA-TPGS-Tf due to transferrin receptor mediated endocytosis mechanism (Fig.5 and

5

Fig.6). In order to demonstrate the advantages of the DTX loaded polymeric micelles, a

6

quantitative index of inhibitory concentration, IC50, which is the drug concentration required to

7

induce 50% death of the cells incubated in a designated period, was determined. For instance, after

8

24 h incubation, IC50 for each of the three types of DTX formulations, namely the

9

DTX-PLA-TPGS and DTX-PLA-TPGS-Tf vs DocelTM were determined from the cytotoxicity

10

data to be 5.12 ± 1.39, 0.63 ± 0.22 vs 45.98 ± 1.70 µg/mL, respectively observed for A549 cells

11

which imply that the drug formulated in the DTX-PLA-TPGS and DTX-PLA-TPGS-Tf could be

12

7.7 and 70.34 folds more efficient than DocelTM after 24 h treatment (Table 3), respectively. Also,

13

results indicate that in order to kill A549 cells, the DTX-PLA-TPGS and DTX-PLA-TPGS-Tf

14

require significantly (P<0.05) lower drug concentrations in comparison to DocelTM (Fig.5 and

15

Table 4). DTX-PLA-TPGS has enhanced cytotoxicity in A549 cells because of PLA-TPGS

16

amphiphilic structure polymeric micelles, which inhibited P-gp efflux mechanism and provide

17

higher

18

(DTX-PLA-TPGS-Tf) have further enhanced cytotoxicity in A549 cells can be possibly attributed

19

to transferrin receptor targeting effect on the A549 cells (Fig.6). These results suggest that

20

transferrin decoration could significantly increase the cellular uptake of the polymeric micelles

21

[42]. Most importantly, transferrin conjugation on the polymeric micelles (DTX-PLA-TPGS-Tf)

22

would imply high concentration of drug to lung cancer cells which may reduce the systemic side

23

effects as well as cost for clinical use. Fig. 6 shows the cellular uptake mechanism of DTX

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drug

concentration

into

cells

(Fig.6).

The

targeted

polymeric

micelles

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 18

Page 18 of 40

formulations. The targeted and non-trageted polymeric micelles have followed transferrin receptor

2

targeted endocytosis and phogocytosis effects, respectively. Kim et al., have proved that

3

polymeric micelles did not produce any toxicity at the concentration 0.0001–1 µg/ml of

4

mPEG-PDLLA copolymer in both cell lines i.e., human breast cancer cell lines (MCF7) and

5

human ovarian cancer cell lines (OVCAR-3) [37]. Recent study, de Melo-Diogo et al., have

6

proved that the biocompatibility of PLA-TPGS copolymer on 549 cell line, suggested that the

7

PLA-TPGS will not produce cytotoxic effects on cells [17].

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Table 3 (approximate position)

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Figure 5 (approximate position)

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Figure 6 (approximate position) 3.7. In-vivo safety using bronchoalveolar lavage (BAL) fluids of rats

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Lung toxicity of DTX has been reported by various researcher in both mouse and rat

13

models. Normal saline exposure did not show any changes of any biochemical parameters in BAL

14

fluid. The administration of DTX formulation into the animal models induced inflammations and

15

fibrosis. To prove the safety of the DTX for lung cancer therapy, lung toxicity parameters were

16

measured. The lung toxicity parameters were observed after 7, 15 and 30 days of i.v.

17

administrations of PLA-TPGS (placebo micelles), DTX-PLA-TPGS, DTX-PLA-TPGS-Tf and

18

DocelTM. The PLA-TPGS micelles, DTX-PLA-TPGS and DTX-PLA-TPGS-Tf did not induce

19

significant (P>0.05) lung toxicity in comparison to DocelTM treated group, as evidenced from the

20

different pulmonary toxicity marker levels such as ALP, LDH and total protein counts in BAL

21

fluid of rats [50, 51].

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After 7 days ALP, LHD and protein counts levels in BAL fluids were significantly

23

(P<0.05) increased in the animal group which received DocelTM in compared to the control group

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Page 19 of 40

(normal saline treated) of animals (Fig. 7). At 15 and 30 days of post exposure, similar trends were

2

found to DocelTM. In comparison to control group (saline treated), a non-significant (P>0.05)

3

differences were observed in the ALP (Fig.7A), LDH levels (Fig.7B) and total protein counts

4

(Fig.7C) after i.v. administration of DTX-PLA-TPGS and DTX-PLA-TPGS-Tf. The major

5

adverse effects of DTX are their capabilities of damage alveoli cells, tissue damage and induce the

6

pulmonary toxicity after the i.v. administrations. The pulmonary toxicity of DTX is mainly due to

7

the poor solubility in aqueous media and also higher risk to normal cells by the activation of ROS

8

generation in the lung cells (Fig.7).

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Figure 7 (approximate position)

12

4. Conclusions

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In this work, we developed transferrin conjugated PLA-TPGS micelles as effective and

14

safer platforms in DTX delivery for lung cancer therapy. Our research confirms that the solvent

15

displacement method as suitable for loading of DTX into polymeric micelles. The non-targeted

16

and targeted (transferrin conjugated) polymeric micelles showed drug encapsulation efficiency up

17

to 86%. The particle size analysis showed that the polymeric micelles are well structured in nano

18

size between 84.9 to 184.8 nm. In-vitro drug release profiles revealed a desired sustained drug

19

release manner of the polymeric micelles. The DTX-PLA-TPGS and DTX-PLA-TPGS-Tf

20

achieved up to 7.7 and 70.34 folds decrease in IC50 value compared with that of DocelTM after 24

21

h incubation with A549 human lung cancer cell lines. In comparison with the DocelTM, DTX

22

loaded PLA-TPGS micelles were showed significantly higher cytotoxicity, low toxic profiles and

23

thereby improved efficacy as well as safety for lung cancer therapy.

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Page 20 of 40

1 2

Acknowledgements Author Mr. Rahul Pratap Singh acknowledges the University Grants Commission (UGC),

4

New Delhi, India, for the award of Rajiv Gandhi National Fellowship (RGNF)

5

(RGNF-2012-13-SC-UTT-18279) to his doctoral research in the year of 2012-2013.

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Declaration of interest: The authors report no declaration of interest.

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polymer micelles, Cancer Sci. 100 (2009) 572–579.

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(lactide)-vitamin E TPGS nanoparticles, Biomaterials 29 (2008) 2663-2672.

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Biomaterials 32 (2011) 5148-5157.

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nanoparticles as MRI contrast agent, Biomaterials 31 (2010) 5588-5597.

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dots loaded in poly (lactide)- vitamin E TPGS nanoparticles for cellular and molecular imaging,

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with Leu-7 and OKT-9 monoclonal antibodies, Jpn. J. Clin. Oncol. 15 (1985) 537-544.

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nanoparticles, Biomaterials 27 (2006) 4025-4033.

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chemotherapy: synthesis, formulation, characterization and in vitro drug release, Biomaterials 27

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ip t cr us an M d Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Table Captions:

Table 1. Formulae of PLA-TPGS micelles.

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 28

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Table 2. Particle size, polydispersity, zeta potential, encapsulation efficiency and drug loading of DTX loaded polymeric micelles.

d

M

an

us

cr

ip t

Table 3. IC50 values of DTX formulated as PLA-TPGS polymeric micelles or DocelTM for human lung cancer cells (n=4).

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Figure Captions: Fig.1. Schematic diagram for (A) synthesis of (i) PLA-TPGS copolymer (B) activation of (i) TPGS (TPGS-COOH) (ii) synthesis of TPGS-Tf (C) preparation of (i) DTX-PLA-TPGS: DTX loaded polymeric micelles and (ii DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles. Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 29

Page 29 of 40

Fig.2. (A) FTIR spectra of L-LA, TPGS and PLA-TPGS copolymer (B) TPGS-COOH (carboxylated TPGS) and TPGS-Tf (transferrin conjugated TPGS).

cr

ip t

Fig.3. (A) Particle size and (B) polydispersity index of placebo polymeric micelles (PLA-TPGS), DTX-PLA-TPGS: DTX loaded polymeric micelles and DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles and (C) particle size distribution of DTX-PLA-TPGS: DTX loaded polymeric micelles and (D), drug encapsulation efficiency of DTX-PLA-TPGS: DTX loaded polymeric micelles and DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles.

us

Fig.4. In-vitro drug release from DTX loaded preparations in PBS (pH 7.4). Bar represent ± S.D (n=3). DocelTM: commercial DTX preparation (control), DTX-PLA-TPGS: DTX loaded polymeric micelles and DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles.

M

an

Fig.5. Cytotoxicity and antiproliferative profile of DTX formulated as polymeric micelles or DocelTM for human lung cancer cells (n=4). DocelTM (DTX injection), DTX-PLA-TPGS (DTX loaded polymeric micelles) and DTX-PLA-TPGS-Tf (DTX loaded transferrin conjugated polymeric micelles).

d

Fig.6. The proposed mechanisms: passive diffusion, transferrin receptor mediated endocytosis and phagocytosis of various formulations. (A) Passive diffusion of DocelTM (marketed preparation) with p-gp efflux (B) transferrin receptor mediated endocytosis for DTX-PLA-TPGS polymeric micelles, involves the formation of vesicles transferrin receptor regions of the plasma membrane. The transferrin in the cytosol are then recycled back to the plasma membrane followed by movement of ingested materials from early endosome to the late endosome, finally fusing lysosome to form the lysosome-endosome hybrid and (C) phagocytosis occurs in phagosomes for DTX-PLA-TPGS (released DTX was involved in interaction with microtubule).

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Fig.7. In-vivo safety study of DTX loaded TPGS polymeric micelles; (A) Alkaline phosphatase activity (KA Units); (B) LDH activity (U/L) and (C) Total protein counts (g/dl) in BAL fluid of rats. Control; Saline treatment; DocelTM: DTX injection, PLA-TPGS: Placebo polymeric micelles, DTX-PLA-TPGS: DTX loaded polymeric micelles and DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles.

Table 1 Formulae of PLA-TPGS micelles. S. No. 1 2 3

Batch PLA-TPGS (Placebo) DTX-PLA-TPGS DTX-PLA-TPGS-Tf

DTX (mg) 3 3

PLA-TPGS (mg) 25 25 25

TPGS-Tf (mg) 10

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1 PLA-TPGS: Placebo polymeric micelles

3

DTX-PLA-TPGS: DTX loaded polymeric micelles

4

DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles

cr us an M d Ac ce pt e

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

ip t

2

Table 2 Particle size, polydispersity, zeta potential, encapsulation efficiency and drug loading of DTX loaded polymeric micelles. Batches

Particle size (nm)

Polydispersity (mean ± S.Da)

Zeta potential

Encapsulation efficiency

Drug loading

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 31

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(mean ± S.Da) PLA-TPGS DTX-PLA-TPGS DTX-PLA-TPGS-Tf

84.09 ± 7.3 135.3 ± 6.54 184.8 ± 9.22

0.45 ± 0.04 0.40 ± 0.05 0.43 ± 0.04

(mV) (mean ± S.Da) - 12.1 ± 1.8 - 8. 64 ± 0.6 2.88 ± 0.4

71.14 ± 6.7 86.63 ± 4.2

(µg/mg) (mean ± SDa,*) 37.23 ± 3.2 39.69 ± 2.4

ip t

1

(%) (mean ± S.Da,b)

a

n=3

3

b

Drug encapsulation efficiency (%) = (amount of DTX encapsulated in the micelles/ amount of

cr

2

DTX added during micelles fabrication) x 100

us

4

* Drug loading = (weight of DTX (µg) per one mg of the drug-loaded micelles)

6

S.D: Standard deviation

7

PLA-TPGS: Placebo polymeric micelles

8

DTX-PLA-TPGS: DTX loaded polymeric micelles

9

DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles

M

d Ac ce pt e

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

an

5

Table 3 IC50 values of DTX formulated as PLA-TPGS polymeric micelles or DocelTM for human lung cancer cells (n=4). Formulation TM

Docel

IC50 in µg/ml on human lung cancer cells (A549) 45.98 ± 1.70

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 32

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5.12 ± 1.39* 0.63 ± 0.22*,a

DTX-PLA-TPGS DTX-PLA-TPGS-Tf 2

a

n=4

3

*

represents significant differences (p< 0.05) from the DocelTM

4

a

represents significant differences (p< 0.05) from the DTX-PLA-TPGS

5

ip t

1

DocelTM: (DTX injection)

7

DTX-PLA-TPGS: DTX loaded polymeric micelles

8

DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles

us an

9 10 11 12 13 14

M

15

Ac ce pt e

d

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

A

Sn(Oct)2

(i)

+ L-LA

32 33

cr

6

TPGS

120 0C / 4 h, under N2 atmosphere

PLA-TPGS

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 33

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Succinic Anhydride

- COOH

Transferrin - NH2

(ii)

- COOH

NHS/EDC

TPGS-Tf

(i)

+

Docetaxel

+

Ac ce pt e

(ii)

PLA-TPGS

TPGS-Tf

Non-targeted micelles

d

M

PLA-TPGS

an

C

us

TPGS-COOH

1 2

3 4 5 6 7 8 9 10 11

TPGS-COOH

ip t

DMAP / 100 0C

TPGS

cr

B

(i)

Docetaxel

Targeted micelles

Fig.1. Schematic diagram for (A) synthesis of (i) PLA-TPGS copolymer (B) activation of (i) TPGS (TPGS-COOH) (ii) synthesis of TPGS-Tf (C) preparation of (i) DTX-PLA-TPGS: DTX loaded polymeric micelles and (ii) DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles.

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 34

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A

110

TPGS-COOH TPGS-Tf

L -L A T PG S PL A -T P G S

110

90

90

80 70 60 50 40 30

80 70 60 50 40

20 10

30

0 500

100 0

1500

2000

25 00

300 0

3500

4 000

450 0

0

500

1000

1500

2500

3000

3500

4000

4500

Wavenumber (cm-1)

us

W a ven um ber (cm -1 )

d

M

an

Fig.2. (A) FTIR spectra of L-LA, TPGS and PLA-TPGS copolymer (B) TPGS-COOH (carboxylated TPGS) and TPGS-Tf (transferrin conjugated TPGS).

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

2000

cr

0

ip t

Transmittance (%)

T ran sm ittan ce (% )

B

100

100

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 35

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250

0.6

A

150

PLA-TPGS DTX-PLA-TPGS

100

DTX-PLA-TPGS-Tf

Polydispersity index

200

50

0.4 PLA-TPGS

0.3

DTX-PLA-TPGS DTX-PLA-TPGS-Tf

0.2 0.1

0

0 PLA-TPGS

DTX-PLA-TPGS

DTX-PLA-TPGS-Tf

PLA-TPGS

Formulation code

DTX-PLA-TPGS

DTX-PLA-TPGS-Tf

Formulation code

C D

100

cr

80 70 60 50 40 30 20 10

an

0

us

Drug encapsulation efficiency (%)

90

DTX-PLA-TPGS DTX-PLA-TPGS-Tf

DTX-PLA-TPGS-Tf

Formulation code

M

DTX-PLA-TPGS

d

Fig.3. (A) Particle size and (B) polydispersity index of placebo polymeric micelles (PLA-TPGS), DTX-PLA-TPGS: DTX loaded polymeric micelles and DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles and (C) particle size distribution of DTX-PLA-TPGS: DTX loaded polymeric micelles and (D), drug encapsulation efficiency of DTX-PLA-TPGS: DTX loaded polymeric micelles and DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles.

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13

ip t

Particle size (nm)

B

0.5

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 36

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120

ip t

80 DocelTM

60

DTX-PLA-TPGS

DTX-PLA-TPGS-Tf

cr

40

20

0 20

30

40 50 Time (h)

60

70

80

d

M

Fig.4. In-vitro drug release from DTX loaded preparations in PBS (pH 7.4). Bar represent ± S.D (n=3). DocelTM: commercial DTX preparation (control), DTX-PLA-TPGS: DTX loaded polymeric micelles and DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles.

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12

10

an

0

us

Drug released (%)

100

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 37

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100 90

DocelTM DTX-PLA-TPGS DTX-PLA-TPGS-Tf

70

ip t

60 50 40 30

cr

Cell viability (%)

80

20

0 0.025

0.25

2.5

us

10 25

d

M

Fig.5. Cytotoxicity and antiproliferative profile of DTX formulated as polymeric micelles or DocelTM for human lung cancer cells (n=4). DocelTM (DTX injection), DTX-PLA-TPGS (DTX loaded polymeric micelles) and DTX-PLA-TPGS-Tf (DTX loaded transferrin conjugated polymeric micelles).

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

an

Concentration of DTX in PLA-TPGS micelles (µg/ml)

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 38

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A

B

Transferrin

Passive diffusion DocelTM

Transferrin receptor mediated endocytosis DTX-PLA-TPGS-Tf Transferrin receptor

C Phagocytosis DTX-PLA-TPGS

cr

Phagosome

P-gp excretion without inhibition Early endosome

us

Late endosome

ip t

Receptor recycling

Free TPGS

an

Inhibit P-gp

d

Fig.6. The proposed mechanisms: passive diffusion, transferrin receptor mediated endocytosis and phagocytosis of various formulations. (A) Passive diffusion of DocelTM (marketed preparation) with p-gp efflux (B) transferrin receptor mediated endocytosis for DTX-PLA-TPGS polymeric micelles, involves the formation of vesicles transferrin receptor regions of the plasma membrane. The transferrin in the cytosol are then recycled back to the plasma membrane followed by movement of ingested materials from early endosome to the late endosome, finally fusing lysosome to form the lysosome-endosome hybrid and (C) phagocytosis occurs in phagosomes for DTX-PLA-TPGS (released DTX was involved in interaction with microtubule).

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

M

Stabilized microtubule

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 39

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Contol

A

DocelTM

16 )s it n U14 A K ( yt 12 i ivt c 10 A se ta a 8 h p s o h 6 P e in l 4 a lk A 2

PLA-TPGS DTX-PLA-TPGS

ip t

DTX-PLA-TPGS-Tf

0 15 days

B

C

Contol DocelTM

700

12

DTX-PLA-TPGS

600

DTX-PLA-TPGS-Tf

l) /d 8 g( ts n u o C 6 n eit or lP 4 ta o T

500 400 300 200

DocelTM PLA-TPGS DTX-PLA-TPGS

2

100 0 15 days

30 days

M

0

7 days

7 days

15 days

30 days

d

Fig.7. In-vivo safety study of DTX loaded polymeric micelles; (A) Alkaline phosphatase activity (KA Units); (B) LDH activity (U/L) and (C) Total protein counts (g/dl) in BAL fluid of rats. Control; Saline treatment; DocelTM: DTX injection, PLA-TPGS: Placebo polymeric micelles, DTX-PLA-TPGS: DTX loaded polymeric micelles and DTX-PLA-TPGS-Tf: DTX loaded transferrin conjugated polymeric micelles.

Ac ce pt e

1 2 3 4 5 6 7 8 9 10

10

us

PLA-TPGS

Contol

an

) /L U ( yt i ivt c A sea n e g or d y h e D et tac a L

30 days

cr

7 days

Singh et al: Revised submission to Int. Journal of Biological Macromolecules (26 Nov 2015) 40

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