ATRP Fabricated and Short Chain Polyethylenimine Grafted Redox

Subscriber access provided by University of Florida | Smathers Libraries

Article

ATRP fabricated and short chain polyethyleneimine grafted redox sensitive polymeric nanoparticles for codelivery of anticancer drug and siRNA in cancer therapy Chetan Nehate, Aji Alex Moothedathu Raynold, and Veena Koul ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11716 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

ATRP fabricated and short chain polyethyleneimine grafted redox sensitive polymeric nanoparticles for co-delivery of anticancer drug and siRNA in cancer therapy

Chetan Nehateab, Aji Alex Moothedathu Raynoldab, Veena Koulab* a

Centre for Biomedical Engineering, Indian Institute of Technology Delhi, New Delhi 110016, India. b

Biomedical Engineering Unit, All India Institute of Medical Sciences, New Delhi 110029, India. *Corresponding Author Tel: +91 (11) 2659 1041 Email: [email protected]

Keywords: siRNA delivery, nanoparticles, polyplexes, redox responsiveness, pH sensitivity, doxorubicin, polycaprolactone.

1 ACS Paragon Plus Environment


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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 43

ABSTRACT To overcome the limitations of conventional chemotherapy, nanoparticle mediated combinatorial delivery of siRNA and drug represents a new approach to overcome its associated side effects. Designing safe and efficient vehicles for their co-delivery has emerged as a potential challenge in the clinical translation of these formulations. Herein, we have demonstrated a novel “two in one” polyplex nanosystem developed from redox sensitive, short chain

polyethyleneimine

modified

poly[(poly(ethylene)glycol

methacrylate]-s-s-

polycaprolactone copolymer synthesised by ATRP, which can deliver doxorubicin and polo like kinase I siRNA, simultaneously for enhanced chemotherapeutic effect. The nanoparticles were found to be stable at physiological buffer with and without FBS. The developed polymeric nanosystem was found to be biocompatible and hemocompatible in vitro and in vivo at repeated dose administration. The polymer could easily self-assemble into ~100 nm spherical nanoparticles with enhanced doxorubicin loading (~18%) and effective siRNA complexation at polymer to siRNA weight ratio of 15. The doxorubicin loaded nanoparticles exhibited ~ 4 fold higher drug release in endosomal pH (pH 5) containing 10 mmol GSH compared to pH 7.4, depicting their redox sensitive behavior. The polyplexes were capable of delivering both cargos simultaneously to cancer cells in vitro as observed by their excellent co-localization in the cytoplasm of MDA-MB-231 and HeLa cells using confocal laser microscopy. Moreover, in vitro transfection of the cells with polyplexes exhibited 50-70 % knockdown of plk1 mRNA expression in both cell lines. In vivo administration of the drug loaded polyplexes to EAT tumor bearing Swiss albino mice depicted ~ 29 fold decrease in percent tumor volume in comparison to control group. The results highlight the therapeutic potential of the polyplexes as combined delivery of doxorubicin and plk1 siRNA in cancer therapy.


Page 3 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION RNA interference (RNAi) mechanism discovered by Fire, Mello and co-workers in late 1990s 1

, provided an attractive and well documented approach for sequence specific suppression of

genes and has great potential for treatment of various diseases including cancer.2-3 Owing to fast degradation of siRNA in the physiological environment and poor cellular uptake, delivery of siRNA remains a most significant challenge for its therapeutic applications. In clinical status, there are eleven formulations based on RNAi therapeutics, out of these, seven formulations are lipid based, two formulations are based on ex vivo transfection while remaining two are polymer based formulations.4-5 Delivery of siRNA with lipid based nanocarriers is highly efficient, but their clinical use is limited by their toxic nature, particularly during systemic administration in high doses (which is the main cause of their terminations from the clinical state).6 Currently, delivery of siRNA through various biocompatible and biodegradable polymer systems is becoming safer approach for RNAi therapy.7-8 These polymeric nanocarriers form stable nanocomplexes through electrostatic interactions protecting complexed siRNA from nucleases and facilitating its cellular uptake in tumor cells. Polymeric nanoplatforms meet the ideal features for siRNA delivery, because of their excellent safety, good stability in biological fluids and ability to continuously activate RNAi with high efficiency. The simultaneous delivery of chemotherapeutic drug and siRNA has been proven to be a better therapeutic strategy than administering the active agents alone. Many studies have demonstrated that co-delivery of drug and siRNA could achieve synergistic anticancer effect concomitantly reducing the dose and side effects of chemotherapeutic drug.9-10 Doxorubicin (DOX) is a potent anticancer drug which is widely employed in chemotherapy but suffers from major drawbacks of cardiotoxicity and multidrug resistance.11 Current research is being focused to effectively co-deliver DOX and siRNA in order to reduce the dose of DOX and achieving better therapeutic effect.12-14 Over the past decade, significant research is being carried out in developing glutathione (GSH) triggered degradable polymers.15 The stimuli based degradation at elevated levels of GSH in the intracellular environment is triggered by thiol-disulfide exchange reaction, wherein the polymer can be degraded into smaller fragments by replacing disulfide bond with a small thiol group.16 It has been postulated that the cancer cells have high redox potential and therefore depict high levels of GSH (100-1000 times higher, 1-10 mmol) compared to blood and extracellular milieu (2-20 μmol).17 Moreover, tumor tissues also exhibit high hypoxic level



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 43

with at least 4 fold higher GSH level than normal tissue.18 Thus this drastic change of GSH level in tumor cells opens new arena to develop GSH triggered intelligent polymeric nanosystems, thereby promoting more specific disposition of the therapeutic payloads to tumor cells.19-21 Our research group has extensively studied redox sensitive polymeric systems for drug delivery applications.22-26 However, the present work emphasizes on co-delivery of doxorubicin and polo like kinase 1 (plk1) siRNA using redox responsive polymeric nanosystem. Polo like kinases plays a key role in the mitosis of mammalian cells. The plk1 is overexpressed in many cancers and thereby provide a hitting target for gene silencing therapeutics.27 Moreover, the plk1 lipid based therapeutic form Tekmira Pharmaceuticals Corporation is already reached to phase II in a clinical trial for the treatment of neuroendocrine and adrenal tumors.28 Over the past two decades, amphiphilic block copolymers got extensive attention for developing polymeric nanoparticles, resulting in their successful entry in clinical trials29-30 or obtaining clinical approval (i.e. Genexol® PM, Samyang Genex Co., Seoul, Korea). Polycaprolactone (PCL), polylactide (PLA), polyglycolide (PGA), polyethylene glycol (PEG) are highly biocompatible and biodegradable polymers. Herein, we have developed short chain polyethyleneimine (PEI) modified redox sensitive, atom transfer free radical polymerization (ATRP) based poly(polyethylene glycol) methacrylate polycaprolactone copolymer with disulfide linkage. These small PEI (600 ~ Da) and PEG (~ 360 Da) hydrophilic molecules have high water solubility and thereby can be easily eliminated form systemic circulation. Furthermore, multiple PEG chains in polymer provide “stealth nature” to nanoparticles, while short PEI chains offer positive charge over their surface for effective siRNA complexation. It has been reported that over the prolonged administration, high molecular weight polyethylene glycols (non-biodegradable polyethers) can get accumulated in body and may evoke immune response.31 In addition, there are several reports demonstrating, high molecular weight PEI (~ 25000 Da), which is generally used for siRNA delivery to be toxic and non-biodegradable.3233

The use of short chain PEI could effectively overcome this obstacle.34-35 The developed

amphiphilic polymer can be easily self-assembled in aqueous phase providing stable and monodispersed nanoparticles. The developed nanoparticles sensitiveness to high GSH concentration shall be able to shed off the short chains of PEG and PEI, more specifically in tumor cells. As per best of our knowledge, this is the first study validating the use of GSH triggered sheddable “two in one” nanoparticles developed from polycaprolactone with short chains of PEG and PEI for combined delivery of drug and siRNA. To evaluate the efficacy of developed 4 ACS Paragon Plus Environment

Page 5 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


amphiphilic nanocarriers in vitro and in vivo, we have used DOX and polo like kinase 1 siRNA (plk1-siRNA) as a model drug and nucleic acid, respectively. The biocompatibility of developed polymeric nanosystem was evaluated using hemolysis, protein adsorption, coagulation studies, and MTT cytotoxicity assay. Stability study for nanocarriers was conducted in different media. The in vitro drug release was executed at pH 7.4 and pH 5, with and without 10 mmol GSH, to evaluate the impact of redox condition on DOX release behavior from DOX loaded nanoparticles. The in vitro studies in cancer cell lines, MDA-MB-231 and HeLa were carried out for evaluating the cellular uptake and cytotoxicity of DOX loaded nanoparticles bearing siRNA. In vivo biocompatibility and tumor regression potential of the nanoformulation was studied with Ehrlich ascites tumor (EAT) model. Histopathology and serum biochemical parameters were also studied to evaluate the toxicity of the nanosystem.

2. MATERIALS AND METHODS Poly(ethylene glycol) methacrylate (PEGMA) (Mn ~ 360 Da), ε-Caprolactone, 2-hydroxyethyl disulfide, tin (II) octoate, polyethyleneimine (PEI) branched (Mn ~ 600 Da), 2,4,6Trinitrobenzenesulfonic acid (TNBS) was obtained from Alfa Aesar U.K. Polystyrene standards were procured from Waters, U.S.A. Copper (I) bromide (CuBr), N′′pentamethyldiethylenetriamine (PMDETA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT), glutathione (GSH), bovine serum albumin (BSA), were procured from SigmaAldrich (St. Louis, MO, U.S.A.). Doxorubicin was acquired as a gift test sample from Ranbaxy Laboratories, India. Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin solution, fetal bovine serum (FBS), Syber green I, phosphate buffer saline (PBS) powder, DNase I RNase-free (supplied with MnCl2), validated silencer select polo like kinase I siRNA (plk1-siRNA), scrambled siRNA (scr-siRNA, negative control siRNA) and FAM labeled negative control siRNA (FAM-siRNA), TRIzol™ reagent, forward and reverse primers for plk1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were procured from Thermo Fischer scientific, U.S.A. Dialysis membrane (3.5 kDa) was obtained from spectrum labs, U.S.A. Fluoroshield Mounting Medium with DAPI was purchased from Abcam U.S.A. Silica for column chromatography 60-120 mesh size, superior grade agarose with low EEO (electroendosmosis), Tris-acetate-EDTA (TAE) buffer, hematoxylin and eosin staining solutions, triethyl amine was obtained from SISCO research laboratories (SRL), India. Sodium metal, sodium hydrogen carbonate, sodium hydroxide, anhydrous sodium sulfate, dry toluene,



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 43

dimethyl sulfoxide (DMSO), chloroform, dichloromethane (DCM), tetrahydrofuran (THF), methanol, ethyl acetate, hexane from petroleum, diethyl ether, dimethyl sulfoxide-d6, chloroform-d was acquired from (Merck Millipore, MA, U.S.A.). iScriptTM cDNA synthesis kit and SsoFast Evagreen supermix were purchased from BIO-RAD, U.S.A. Ultrapure water with resistivity 18 MΩ cm was procured from Milli-Q system (Merck Millipore, MA, U.S.A.). RNase free water (non DEPC treated, AmbionTM) was used for all studies involving siRNA. 2.1. Synthesis of short chain polyethyleneimine modified, glycol)methacrylate]-s-s-polycaprolactone diblock copolymer

poly[poly(ethylene

The cationic polymer was synthesized sequentially in a step by step manner. In the first step, 2-((2-hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate was used as an initiator for ring opening polymerization of ε-caprolactone to yield PCL macroinitiator. The macroinitiator was further utilized for ATRP reaction with PEGMA to get poly[poly(ethylene glycol)methacrylate]-s-s-polycaprolactone diblock copolymer. Furthermore, hydroxyl groups of polymer were converted into carboxyl groups by reacting with succinic anhydride and further modified with short chains of polyethyleneimine (Scheme 1).


Page 7 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1: Synthesis scheme of short chain polyethyleneimine modified, poly[poly(ethylene glycol)methacrylate]-s-s-polycaprolactone copolymer.

2.1.1. Synthesis of 2-((2-Hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate The 2-((2-Hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate was synthesized as per our published work.22 The obtained purified product was characterized using 1H NMR, 13C NMR and high resolution mass spectroscopy (HRMS). Yield = 48.90 %. 2.1.2. Synthesis of bromine terminated polycaprolactone macroinitiator (Br-ss-PCL-OH) Polycaprolactone macroinitiator with disulfide bond was synthesized by ring 2-((2Hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl

propanoate

mediated

ring

opening

polymerization of ε-caprolactone in which tin(II) octoate was used as a catalyst. 2-((2Hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate (200 mg, 0.658 mmol), εcaprolactone (6.57 g, 57.640 mmol, initiator/monomer ratio 1:87.598) were solubilized in 10 mL of dry toluene in the Schlenk flask. Nitrogen gas was purged for three times for 5 min over 7 ACS Paragon Plus Environment


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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 43

the period of 30 min. The catalyst tin (II) octoate (5 mol % of initiator, 10.653 μL, 0.032 mmol) was added to the solution, and the polymerization reaction was carried out at 110 °C for 24 h under N2 atmosphere. After 24 h, the viscous crude product was removed and toluene was evaporated on a rotavapor. The pure product was obtained by precipitation of crude product thrice in cold methanol. Yield 89 %. The polymer was characterized with 1H NMR and

13

C

NMR (Bruker, U.S.A.), FTIR (Bruker, U.S.A.), and gel permeation chromatography (GPC) (Waters, U.S.A.). GPC analysis was performed based on the parameters reported in our previous work.23 2.1.3. Synthesis of poly[poly(ethylene glycol) methacrylate]-polycaprolactone diblock copolymer by atom transfer radical polymerization (ATRP) [(PEGMA)n-ss-PCL-OH] Weighed amounts of Copper (I) bromide (CuBr) (51.642 mg; 0.360 mmol) and N′′Pentamethyldiethylenetriamine (PMDETA) (75.166 μL; 0.360 mmol) were added to 10 mL of dry toluene in a Schlenk flask with continues purging of N2 gas for 30 min to form a blue colored complex. PEGMA (Mn = 360 Da, 1.167 mL; 3.57 mmol) and Br-ss-PCL-OH macroinitiator (3.0 g; 0.30 mmol) were mixed with reaction components at 85 ºC for 24 h under consistent N2 environment. PCL macroinitiator (3.0 g; 0.30 mmol) was added to reaction components and further permitted to stir at 85 °C for 24 h under consistent N2 environment. The reaction components, macroinitiator, PMDETA and CuBr were taken in the proportion of 1:1.2:1.2. After 24 h, the metal complex was removed from the product by passing reaction mixture through a basic alumina column using THF as eluent. The THF from eluate was evaporated over a rotavapor, and the gooey crude solution was precipitated three times in frosty methanol. The purified product was dried on rotavapor and characterized by 1H NMR and 13C NMR (Bruker, U.S.A.), FTIR, and GPC. Yield = 76 %. 2.1.4. Conversion of hydroxyl groups of poly[poly(ethylene glycol) methacrylate]polycaprolactone diblock copolymer to carboxyl groups by succinic anhydride [(HOOCPEGMA)n-ss-PCL-COOH] ATRP fabricated (PEGMA)n-ss-PCL-OH diblock copolymer (2 g; 0.143 mmol) was solubilized in 50 mL of dry THF in Schlenk flask. DMAP (209.643 mg; 1.716 mmol) and Succinic anhydride (171 mg; 1.716 mmol) were added to the reaction mixture and continued to stir at 37 °C for 24 h under N2 environment. The pure product was obtained by precipitating three times in cold methanol. The diblock copolymer with carboxyl group functionality was 8 ACS Paragon Plus Environment

Page 9 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


characterized by 1H NMR, 13C NMR, and CHNS analysis (Vario EL III, Elementar, Germany). Yield = 86 %. 2.1.5. Modification of poly[poly(ethylene glycol) methacrylate]-polycaprolactone diblock copolymer with short chain polyethyleneimine [(PEI-PEGMA)n-ss-PCL-PEI] The carboxyl group terminated poly[poly(ethylene glycol) methacrylate]-polycaprolactone diblock polymer (1.0 g; 0.071 mmol) was dissolved in dry DCM (50 mL). EDC (0.133 g; 0.858 mmol) and NHS (98.747 mg; 0.858 mmol) were added to the flask subsequently. After 1 h, PEI (0.514 g, 0.858 mmol) was mixed with the reaction mixture and permitted to stir at 37 °C for 24 h under N2 environment. After 24 h, DCM was evaporated on rotavapor and the crude product was dissolved in DMSO and dialyzed using dialysis bag (MWCO, 3.5 kDa) against distilled water for 48 h. The purified product was further lyophilized and characterized by 1H NMR, 13C NMR, FTIR, PXRD (Philips X'Pert PRO, X-ray diffractometer, U.S.A.), GPC and CHNS analysis. Yield = 84 %.

2.2. Formulation and characterization of nanoparticles Polymeric blank nanoparticles were formulated by nanoprecipitation method. In brief, polymer (30 mg) was dissolved in 1 mL of DMSO. This solution was added dropwise to 20 mL of water (Milli-Q) under continuous stirring for 20 min. Nanoparticles dispersion was dialyzed against distilled water for 24 h using dialysis bag (MWCO, 3.5 kDa) to remove DMSO. Water was changed thrice during the course of dialysis. DOX loaded polymeric nanoparticles (DOXnanoparticles) were prepared in a similar fashion as that of blank nanoparticles except for the addition of doxorubicin HCl (at a various drug to polymer feed ratios ranging from 0.050.5) and trimethylamine (equal molar amount of doxorubicin HCl) in the DMSO. The DMSO and unencapsulated DOX was removed by dialysis against distilled water for 24 h with changing water three times during the course of dialysis process. DOX loading in the nanoparticles was analyzed by UV-visible spectrophotometer. Briefly, the

DOX

nanoparticles

were lyophilized (Labconco, 4.5 L, Cascade bench top freeze dry system, U.S.A.) and dissolved in DMSO. The DOX loading was determined by recording absorbance readings at 481 nm and calculating their respective concentrations using standard calibration curve in DMSO. We have accounted any absorbance due to the polymer at 481 nm, where the polymer absorbance was found to be negligible (Data not shown). Particle size and zeta potential of nanoparticles were measured using dynamic light scattering, DLS and laser doppler



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 43

velocimetry, LDV (Zetasizer Nano ZS, Malvern Instruments Ltd., U.K.). To study the characteristic of DOX in DOX loaded nanoparticles, Powder X-ray Diffraction (PXRD) studies were conducted with DOX, DOX loaded nanoparticles, pure DOX and nanoparticles physical mixture and blank nanoparticles were analyzed for XRD pattern. Measurements were performed on X-ray diffractometer using scan step time of 30 sec with 2θ range of 10-80° in continuous mode at generator settings of 45 kV and 40 mA. The DOX loading and encapsulation efficiency were determined using following formulae, Loading efficiency (%) =

(Weight of DOX loaded in nanoparticles) (Weight of nanoparticles)

× 100

(Amount of DOX loaded in nanoparticles)

Encapsulation efficiency (%) = (Amount of DOX taken for loading in nanoparticles) × 100

2.3. Nanoparticles-blood compatibility Nanoparticles-blood interaction studies were assessed using hemolysis and coagulation study. Human blood was procured from All India Institute of Medical Sciences (AIIMS), New Delhi, India. 2.3.1. Hemolysis study The hemocompatibility of polymeric nanoparticles was assessed by evaluating the hemolytic nature of the nanoparticles in PBS pH 7.4. To study the efficacy of nanoparticles for proton sponge effect, the endosomolytic effect of the nanoparticles was determined by studying the hemolysis in buffers of endolysosomal pH (pH 5.0) and early endosomal pH (pH 6.2) as described in the literature.36-37 The hemolytic behavior of polymeric nanoparticles [(HOOCPEGMA)n-ss-PCL-COOH], which does not contain PEI chains was also evaluated in buffers of pH 5.0 and pH 6.2.

2.3.2. Coagulation studies The polymeric nanoparticles, dispersed in 100 µl of PBS pH 7.4 were incubated in 900 µL of blood in an incubator shaker at 37 °C at 120 rpm for 1 h, so that final concentration will be in the range from 0.1-2 mg/mL. The prothrombin time (PT) and activated partial thromboplastin


Page 11 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


time (aPTT) were determined as per the reported method.37 Control values for PT and aPTT were obtained by using PBS pH 7.4 treated platelet poor plasma as a negative control.38

2.4. Drug release studies The DOX release profile was evaluated in PBS pH 7.4, PBS pH 7.4 with 10 mmol and 20 μmol GSH simulating the reducing conditions of cytoplasm or endolysosomes and blood circulation respectively.17, 39-40 DOX release profile was also evaluated in endosomal pH using citrate buffer pH 5 with and without 10 mmol GSH. Briefly, DOX loaded nanoparticles (DOXnanoparticles) containing 1 mg of DOX were sealed in a dialysis bag (MWCO 3.5 kDa) and dialyzed against respective buffer solutions at 37 ºC and 120 rpm. The 1 mL of buffer solution was taken out at different time intervals and fresh 1 mL of buffer medium was added to simulate sink condition. The collected samples were immediately examined by UV-visible spectrophotometer (LAMBDA 650 UV-vis spectrophotometer, U.S.A.). DOX standard calibration curve was used to calculate drug amount in release samples. 2.5. Preparation of DOX loaded nanoparticles/siRNA polyplexes (DOXpolyplexesscr-siRNA) and gel retardation study

A predetermined amount of scr-siRNA was added to 200 µl micro centrifuge tubes containing appropriate amounts of cationic DOX loaded nanoparticles. The mixture was vortexed for 10 s and then kept still at room temperature for 30 min to facilitate complexation of siRNA on nanoparticles. A series of DOXpolyplexesscr-siRNA formulations were prepared at different weight ratios. To evaluate the binding efficiency of siRNA to polymeric nanoparticles, gel electrophoresis was carried out using 1.2 % (w/v) agarose gel in TAE buffer. In brief, DOX

polyplexesscr-siRNA at DOXnanoparticles/scr-siRNA weight ratios of of 2 : 1, 3.5 : 1, 5 : 1, 6.5

: 1, 8 : 1, 10 : 1, 15 : 1, 20 : 1, 25 : 1, 30 : 1, 40 : 1, 50 : 1 in PBS pH 7.4, were prepared for gel electrophoresis. Electrophoresis was performed for 25 minutes at 70 V using horizontal gel electrophoresis unit (Mini-Sub® Cell GT Cell, Bio Rad, U.S.A.). The bands of siRNA were visualized by immersing agarose gel in syber green I solution for 1 h in dark and visualized under UV illuminator, and images were captured using molecular imager (Gel Doc XR, BIORAD, U.S.A.).26 The composition of nanoparticles and their notations are provided in table 1.



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Morphological characterizations of

Page 12 of 43

DOX

polyplexesscr-siRNA were performed by high-resolution

transmission electron microscope (HRTEM) (Technai G2, 200 kV, FEI, U.S.A.), Environmental scanning electron microscope (FESEM model, FEI Quanta 200F with OxfordEDS system IE 250 X Max 80) and atomic force microscope (AFM; Dimension icon Scan Asyst, Bruker, U.S.A.). Table 1. The composition of nanoparticles with their notation. Nanoparticles notation

Composition

Nanoparticles

Polymeric blank nanoparticles.

DOX

DOX loaded polymeric nanoparticles.

Polyplexesscr-siRNA Polyplexesplk1-siRNA

Polymeric nanoparticles bearing scr-siRNA. Polymeric nanoparticles bearing plk1 siRNA.

DOX

DOX loaded polymeric nanoparticles bearing scr-siRNA.

DOX

DOX loaded polymeric nanoparticles bearing plk1-siRNA.

nanoparticles

polyplexesscr-siRNA polyplexesplk1-siRNA

2.6. Colloidal Stability studies of DOXpolyplexesscr-siRNA The stability of DOXpolyplexesscr-siRNA at a weight ratio of 15, was studied using DLS in different media such as PBS pH 7.4, DMEM supplemented with 10 % FBS, PBS pH 7.4 with 20 μmol GSH and PBS pH 7.4 with 10 mmol GSH for 72 h at 37 °C. Briefly, 125 μL of 800 μg/mL polyplexesscr-siRNA suspension was diluted in 2 mL of corresponding media, at 37 ºC, and

DOX

the nanocarriers’ size and PDI were monitored using DLS at different time intervals for 72 h. The influence of GSH on disulfide bond in polymer backbone was evaluated at 10 mmol and 20 µmol concentration in PBS pH 7.4.25

2.7. Cell Culture Studies

MDA-MB-231 (human breast adenocarcinoma cell line) and HeLa (human cervical cancer cell line) were obtained from NCCS, Pune, India. Cells were grown in T-25 cm2 tissue culture flasks supplemented with cell culture media containing 10 % fetal bovine serum (FBS). Leibovitz's L-15 medium was used for MDA-MB-231, while HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) in 5 % CO2 atmosphere at 37 °C.


Page 13 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.7.1. In vitro cellular uptake and nuclear localization of polyplexes The uptake and sub cellular distribution of DOXpolyplexesFAM-siRNA in MDA-MB-23 and HeLa cells was analysed using confocal laser scanning microscope (CLSM). The cells dispersed in media were seeded on coverslips in 24 well plate at a density of 5 × 104 cells per well and kept in CO2 incubator at 37 °C for 24 h. After reaching 70-80 % confluency, DOXpolyplexesFAM-siRNA with a DOX and FAM-siRNA concentration equivalent to 5 μg/mL and 100 nmol were incubated in cells for 2 h at 37 °C in a CO2 incubator. Cells were rinsed with PBS pH 7.4 thrice and fixed with 4 % paraformaldehyde solution for 20 min. After fixation paraformaldehyde solution was removed and cells were again rinsed with PBS pH 7.4 thrice and stained with DAPI containing a fluoroshield mounting medium. Cells containing coverslips were then seen under CLSM (FluoView FV1000 Olympus, U.S.A.) with respective emission wavelengths for DAPI (460 nm), DOX (560 nm) and FAM-siRNA (488 nm) at 60 × magnification.41 Nuclear localization of DOXpolyplexesFAM-siRNA was also evaluated in aforesaid cell lines in a similar fashion as described above where incubation time of DOXpolyplexesFAM-siRNA was increased up to 8 h.

2.7.2. Quantitative evaluation of in vitro cellular uptake by flow cytometry

The quantitative cellular uptake of

DOX

polyplexesFAM-siRNA in MDA-MB-231 and HeLa cells

was analysed by using flow cytometry. The cells dispersed in media were cultured in a six-well plate at a density of 1 × 105 cells/well and kept in a CO2 incubator at 37 ºC for 24 h. polyplexesFAM-siRNA with a DOX and FAM-siRNA concentration equivalent to 5 μg/mL and

DOX

100 nmol respectively were incubated with cells at 37 ºC for 2 h. After incubation, cells were rinsed thrice with PBS pH 7.4 and resuspended in PBS pH 7.4 after trypsinization. The cell pellet was obtained by centrifugation at 3500 rpm for 5 min. The pellet was dispersed in PBS pH 7.4 and analysed using a flow cytometer (BD accuri C6, U.S.A.).

2.7.3. Real time polymerase chain reaction (RT-PCR) analysis

MDA-MB-231 and HeLa cells dispersed in culture media were cultured in six well plates and kept in a CO2 incubator at 37 ºC. After reaching the 70-80 % confluency, cells were incubated



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 43

with polyplexesplk1-siRNA and polyplexesscr-siRNA formulations at different concentrations of plk1-siRNA (50, 100 and 125 nmol) for 48 h at 37 °C and 5 % CO2. Cells were harvested by trypsin solution for RNA isolation. RNA was isolated using TRIzol reagent. The complementary DNA (cDNA) was prepared as per given protocol using iScriptTM cDNA synthesis kit, BIO-RAD. Briefly, isolated RNA solutions were mixed with cDNA reaction buffer, DNAase I and reverse transcriptase enzymes. Nuclease-free water was added to each sample to adjust reaction volume to 10 µL so that final concentration of cDNA reaction buffer become 1X. Three PCR cycles were executed (25 ºC for 5 min, 46 ºC for 20 min and 95 ºC for 1 min) using PCR analyzer (CFX96TM Real-Time System, BIO-RAD, U.S.A.). The prepared cDNA was stored at 4 ºC for further use. The cDNA samples were mixed with forward and reverse primers and SsoFast Evagreen supermix, BIO-RAD. The final reaction volume was kept at 20 µL by adding nuclease-free water. The relative gene expression values were determined by ΔΔCT method, where plk1 gene expression was normalized with housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the endogenous reference and relative to the untreated control cells. The primer sequences for plk1 and GAPDH used for the study are as follows, plk1˗1 forward: reverse:

5’˗CCCATCTTCTGGGTCAGCAAG-3’, plk1˗1

5ʾ˗AAGAGCACCCCCACGCTGTT-3’,

5ʾ˗TGCACCACCAACTGCTTAGC-3’,

GAPDH GAPDH

forward: reverse:

5ʾ˗GGCATGGACTGTGGTCATGAG-3’

2.7.4. Cell viability The cytotoxicity of blank nanoparticles, DOXpolyplexesscr-siRNA and DOXpolyplexesplk1-siRNA were evaluated in MD-MB-231 and HeLa cell lines using MTT assay. In brief, 5 × 103 cells dispersed in media were seeded per well in a 96-well plate and kept in a CO2 incubator at 37 °C. After reaching 70-80 % confluency, blank nanoparticles at 0.1-2 mg/mL concentration range were incubated with cells at 37 ºC for 48 h. Further to address the synergistic effect of DOX and siRNA,

DOX

polyplexesscr-siRNA and

DOX

polyplexesplk1-siRNA formulations with a final

DOX concentration at 0.05-8 µg/mL and siRNA at 100 nmol concentration were incubated with cells for 48 h. The percent cell viability was calculated as per the reported method.22


Page 15 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.8. Animal studies 2.8.1. In vivo toxicity study of the cationic nanoparticles The 7-8 week old female swiss albino mice (25 ± 5 g) were obtained from the central animal facility, AIIMS, New Delhi, India, and were randomly divided into two groups (n = 6) for evaluating repeated dose toxicity study. The experiments were performed as per the animal ethical guidelines (796/IAEC/14) governed by AIIMS. Blank polymeric nanoparticles dispersed in PBS pH 7.4 (250 μL) were administered in one group (on days 0, 3, 6 and 9) at a dose of 100 mg/kg, intraperitoneally. The second group was injected with blank PBS pH 7.4 (250 μL). On day 11, blood samples were collected from the mice by a retro-orbital venous puncture and hematological and serum biochemical parameters were evaluated.42 The animals were then sacrificed by cervical dislocation and vital organs were collected in 10 % neutral buffered formalin and at kept at 37 ºC. Histopathological evaluation of organs was performed using Hematoxylin-Eosin staining. 2.8.2. In vivo anticancer efficacy 7-8 week old female Swiss albino mice (25 ± 5 g) were housed at 25º C with adequate humidity in polycarbonate cages, with sterile water ad libitum and chow food, throughout the experimental period. Consistent and uninterrupted 12 h light and the dark cycle was maintained. The in vivo antitumor effect of various nano-formulations was assessed using Ehrlich ascites tumor (EAT) model. Tumors were prompted by injecting EAT cells ( ~2 × 107 cells/mice in 150 μL of PBS pH 7.4) over the dorsal side of female Swiss albino mice, subcutaneously.43 The nanoformulation treatment was started at a day assigned as 0, when tumor volume reached about 250-300 mm3. Animals were randomly divided into 5 groups containing 6 animals in each group. Groups were divided for respective formulation such as control i.e. PBS pH 7.4 treated, polyplexesplk1-siRNA treated, DOX treated, DOXpolyplexesscr-siRNA treated and

polyplexesplk1-siRNA treated. All the formulations (50 μl suspension in PBS pH

DOX

7.4) were administered intratumorally in the longitudinal direction from the edge to the center of the tumors. Injections were given four times over a period of 2 weeks (days 0, 3, 6, 9). DOX and siRNA were administered at a dose of 1.5 mg/kg and 0.5 mg/kg respectively (cumulative dose 6 mg/kg and 2 mg/kg respectively). Each injection was administered slowly over 1 minute. Following each injection, the needle was kept at the injection site for further 5 minutes



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 43

in order to prevent any leakage of the sample. Tumor diameters were determined with digital Vernier caliper and tumor volumes were calculated by following formula, Tumor volume = 0.5 × L × W2 Where L denotes the longest tumor diameter, while W, symbolizes the perpendicular diameter. At the end point of the study, animals were sacrificed by cervical dislocation followed by collecting tumors, vital organs, and blood. 2.8.3. Histopathological evaluation of the tumor and organs Histopathological evaluation of tumors and other important organs such as liver, kidneys, heart and lungs was performed to assess the antitumor effect and toxicity profile of the different nanoformulations in mice during the course of tumor inhibition study as per the procedure reported in the literature.37 2.8.4. Analysis of hematology and serum biochemistry parameters The blood was collected from mice in respective groups and hematological parameters were determined. The blood serum samples were analysed for cardiotoxicity assessment using parameters such as creatine kinase MB. Serum samples were also examined for kidney and liver functional efficiency to rule out any toxicity. The serum parameters were measured using fully automated chemistry analyser (TurboChem 100, Awareness Technology, U.S.A.).44 2.9. Statistical Analysis The in vitro data is introduced as means with standard deviation (SD), while in vivo data is communicated as means with standard error mean (SEM). Statistical analysis was calculated by using GraphPad prism (Version 7, GraphPad Software, Inc., La Jolla, U.S.A.). One-way ANOVA was utilized for calculating the level of statistical significance, which is considered at p-value < 0.05.


Page 17 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3. RESULTS AND DISCUSSION 3.1 Preparation of short chain polyethyleneimine modified, poly[poly(ethylene glycol)methacrylate]-s-s-polycaprolactone copolymer Short chain polyethyleneimine modified, poly [poly(ethylene glycol)methacrylate]-s-spolycaprolactone diblock copolymer was developed as depicted in Scheme 1. In the first step, 2-((2-hydroxyethyl)-disulfanyl)ethyl-2-bromo-2-methyl propanoate was synthesized by reacting one equivalent of 2-bromoisbutyryl bromide with 2-hydroxyethyl disulfide. The reaction provided mono bromine functionalized 2-hydroxyethyl disulfide. The mono bromo-functionalized product was purified from impurities such as di-bromo attached 2-hydroxyethyl disulfide and unmodified 2-hydroxyethyl disulfide by silica gel column chromatography. The obtained pure product was further characterized by 1H NMR (Figure S1) and 13C NMR (Figure S2) and HR-MS spectroscopy (Figure S3). In the second step, ATRP macroinitiator (Br-ss-PCL-OH) was synthesized using ROP of ε-Caprolactone with 2-((2-hydroxyethyl)-disulfanyl)ethyl-2-bromo-2-methyl propanoate as an initiator with tin (II) octanoate as a catalyst. The macroinitiator was thoroughly characterized by 1H NMR (Figure S4),

13

C NMR (Figure S5), FTIR (Figure S6), and GPC

(Figure S7). 1H NMR spectrum of purified polymer showed the presence of cystamine side chain with α-bromoisobutyryl bromide group. The multiplet peak at δ 1.336-1.436 conforming to the methylene groups of caprolactone chain (-CO-CH2-CH2-CH2-CH2-CH2-OH), multiplet peak at δ 1.604-1.673 indicating methylene groups of the caprolactone unit (m, -CO-CH2-CH2CH2-CH2-CH2-OH), and triplet peak at δ 2.287-2.337 depicting methylene groups adjacent to carbonyl carbon atom of caprolactone chain (-CO-CH2-CH2-CH2-CH2-CH2-OH), confirmed the existence of polycaprolactone backbone in the developed macroinitiator. The peak at δ 1.945 matches to the methyl groups adjacent to bromine atom of α-bromoisobutyryl bromide, while the triplet peak at δ 2.928 corresponds to the methylene protons next to the disulfide linkage (t, -CH2-CH2-S-S-CH2-CH2-). Thus,

1

H NMR confirmed the formation

polycaprolactone based macroinitiator with disulfide linkage on its side chain. The number average molecular weight (Mn) of macroinitiator based on 1H NMR integration was found to be 10992 Da. As shown in FTIR spectrum, C-Br stretching vibrational band peak at 642 cm-1 also established the successful development of macroinitiator. The synthesized polymer was further characterized by using GPC and number average molecular weight (Mn) was found to be 13699 Da with a polydispersity index (PDI) of 1.73. 17 ACS Paragon Plus Environment


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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 43

In the third step, [(PEGMA)n-ss-PCL-OH] diblock copolymer was synthesized using macroinitiator (Br-ss-PCL-OH) with ATRP reaction. In the presence of N2 environment, the catalyst PMDETA and CuBr allowed the ATRP of PEGMA with macroinitiator to generate amphiphilic diblock copolymer with controlled hydrophobic and hydrophilic ratio in the polymer chain. The diblock ATRP based polymer was further characterized by 1H NMR (Figure S8), 13C NMR (Figure S9), FTIR (Figure S10) and GPC (Figure S11). In 1H NMR, a peak at δ 3.654, conforming to -OCH2 groups of PEGMA chains proved the successful development of the diblock copolymer. The 1H NMR peak integration displayed that 7 molecules of PEGMA have been attached to a polymer chain. The Mn calculated form 1

H NMR was 13500 Da while by GPC, Mn of the polymer was found to be 16292 Da, with a

PDI of 1.43. The

13

C NMR spectrum also confirmed the successful synthesis of the diblock

copolymer. The conjugation of PEGMA chains to diblock copolymer was also confirmed by FTIR spectrum, where the C-O-C vibration band and O-H stretching vibration band was observed at 1100 cm-1 and 3400-3500 cm-1 respectively. In this manner, the copolymer with a preferred molecular weight distribution was developed using ATRP reaction. The hydroxyl groups of multiple PEG chains of ATRP fabricated polymer, [(PEGMA)n-ss-PCL-OH] were further modified to carboxyl groups by reacting the polymer with succinic anhydride. The formation of the carboxylated diblock copolymer was evaluated by 1H NMR (Figure S12), 13C NMR (Figure S13) and GPC (Figure S14). The successful ring opening of succinic anhydride by PEG chains in the polymer was confirmed by 1H NMR depicting a peak at δ 2.629 ppm, conforming to -CH2-CH2- units of succinic acid. Moreover, the 13C NMR also confirmed the successful formation of carboxyl groups terminated diblock copolymer [(HOOC-PEGMA)n-ss-PCL-COOH]. The GPC spectra displayed Mn as 17054 Da with a PDI of 1.70. Carboxyl groups terminated copolymer [(HOOC-PEGMA)n-ss-PCL-COOH] was further modified with short chains of branched, polyethyleneimine (PEI), 600 Da. This low molecular weight PEI was selected because of its relatively less cytotoxicity than high molecular weight PEI (25 kDa).45-47 The multiple carboxyl groups of diblock copolymer were reacted with PEI by EDC, NHS coupling to form short chain PEI functionalized PEGylated amphiphilic copolymer [(PEI-(PEGMA)n-ss-PCL-PEI]. The synthesized amphiphilic copolymer was further characterized by 1H NMR (Figure S15), 13C NMR (Figure S16), FTIR (Figure S17), and GPC (Figure S18). In 1H NMR spectra multiplet peak at δ 2.266 depicts the 18 ACS Paragon Plus Environment

Page 19 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PEI protons. Moreover, the presence of PEI in polymer system was also evaluated by CHNS analysis, where nitrogen content of [(PEI-PEGMA)n-ss-PCL-PEI] polymer was increased to 1.92 ± 0.14 % (Table S1), suggesting successful modification carboxyl group terminated copolymer by PEI. In FTIR, the increase in the broadness of peak between 3300-3500 cm-1 depicts the presence of PEI. The

13

C NMR spectra for [(PEI-PEGMA)n-ss-PCL-PEI] also

corroborates the successful PEI modification on [(HOOC-PEGMA)n-ss-PCL-COOH] diblock copolymer. The GPC for final polymer system depicted Mn as 20116 with PDI of 1.43.

3.2 Formulation and characterization of polymeric nanoparticles Blank polymeric nanoparticles were formulated using nanoprecipitation method as per previously reported procedure.48 The amphiphilic polymer could easily self-assemble into stable polymeric nanoparticles with average particle size of 97.65 ± 6.88 nm and PDI 0.12 ± 0.18, while the zeta potential of blank polymeric nanoparticles was found to be 37.16 ± 1.90 mV (Figure S19 A,C). The primary amine content attributed due to PEI in nanoparticles was determined by TNBS assay and found to be 40.97 ± 1.71 μmol/150 μg of nanoparticles (Supporting information S1). The protein adsorption on blank polymeric nanoparticles was evaluated by BSA adsorption study and found to be 17.20 ± 1.77 % (Supporting information S2). This low protein adsorption on developed nanoparticle formulation was credited to polyethylene glycol chains in nanoparticles. It has been well established that PEG chains in nanoparticles around hydrophobic core assist in minimizing the interaction of proteins with the surface of nanoformulation and thereby providing stability in aqueous media.49 The DOX loaded nanoformulation was optimized based on drug loading content and optimum particle size. Different feed ratios of polymer and DOX were employed for optimization of the formulation (Table 2). The loading efficiency of DOX in nanoparticles was found to be increased with increasing drug/polymer weight ratio. The formulation P3 (Table 2) showed optimum drug loading of 18 ± 1.3 % obtained at drug/polymer weight ratio of 0.25. The size and PDI of this optimized formulation was found to be 108.2 ± 2.2 nm and 0.1 ± 0.1 respectively, while zeta potential was found to be 38.1 ± 1.7 mV. This positive zeta potential must be attributed to PEI chains present at the periphery of nanoparticles. Further increase in theoretical loading of the drug leads to increase in particle size without any significant increase in drug loading. The

DOX

nanoparticles showed a small increase in size as compared to blank

nanoparticles, while zeta potential did not show any significant change (Figure S19 B,D). 19 ACS Paragon Plus Environment


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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 43

To evaluate the physical status of DOX in polymeric nanoparticles, powder X‐ray diffraction (PXRD) study was conducted for DOX, DOXnanoparticles, DOX and nanoparticles as a physical mixture and blank polymeric nanoparticles. The XRD pattern of pure DOX displayed twelve major crystalline peaks at 2θ of 11.78°, 13.28°, 16.83°, 17.63°, 18.53°, 19.53°, 20.78°, 22.73°, 23.58°, 25.23°, 26.48°, 30.33° indicating the crystalline state of DOX (Figure S20). In a physical mixture of DOX and blank polymeric nanoparticles, characteristic peaks of DOX were still observed suggesting a crystalline state of DOX within the physical mixture. While in XRD pattern of

DOX

nanoparticles, diffraction peaks of DOX got vanished

indicating the amorphous state of DOX in nanoparticles. This loss in crystalline nature of DOX in nanoparticles leads to low crystal lattice energy which is required to overcome during drug solubilization. Thus, the amorphous state of DOX in the nanoparticles helps in faster DOX dissolution and release.50 The gel retardation study depicts no binding of scr-siRNA with the polymer at lower weight ratios from 2-8 (Figure S21). Effective complexation of siRNA to polymer was observed from polymer to siRNA weight ratio 10 (Figure 1F). All in vitro studies were performed by considering polymer to scr-siRNA weight ratio as 15, which can provide effective complexation and condensation of siRNA with the polymer. The size and zeta potential were determined for

DOX

polyplexesscr-siRNA using DLS and LDV respectively. The size was found

increased to 135.5 ± 6.3 nm with PDI 0.11 ± 0.1 (Figure 1A), while zeta potential value got reduced to 22.3 ± 0.6 mV (Figure 1B). This increase in size and reduction in zeta potential for polyplexes is attributed to condensation of scr-siRNA over the nanoparticles surface. The morphological characteristics and absolute size of DOXpolyplexesscr-siRNA were determined by TEM (Figure 1C), FESEM (Figure 1D) and AFM (Figure 1E). The absolute size was found to be between 100-120 nm for all three characterization techniques which is in a good agreement with DLS size. Nanoparticles were found to be uniformly spherical in shape without showing any aggregation.


Page 21 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2: DOX loading optimization for different formulations with their size and zeta potentiala. Drug to

Drug

Polymer

Formulation

Zeta Loading

Entrapment

Average Size

efficiency (%)

efficiency (%)

(nm)

polymer

Taken

taken

feed ratio

(mg)

(mg)

P1

0.05

1.5

30

2.7 ± 0.4

46.6 ± 6.4

104.6 ± 1.9

0.07 ± 0.02

31.2 ± 2.6

P2

0.1

3

30

6.9 ± 1.8

56.9 ± 6.1

107 ± 1.6

0.05 ± 0.02

32.3 ± 1.8

P3

0.25

7.5

30

18 ± 1.3

67.7 ± 4.8

108.2 ± 2.2

0.07 ± 0.05

38.1 ± 1.7

P4

0.5

15

30

18.9 ± 0.9

76.6 ± 3.5

360.7 ± 15.3

0.4 ± 0.09

36.6 ± 4.7

code

PDI Average

potential (mV)

a

Mean ± SD, n = 3.



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 43

Figure 1. Dynamic Laser Scattering (DLS) and morphological studies of DOXpolyplexesscr-siRNA at polymer/siRNA weight ratio 15, (A) Size, (B) Apparent zeta potential. (n = 3). Figure (C) HRTEM, scale bar 500 nm, (D) FESEM, and (E) AFM. Figure (F) Represents agarose gel electrophoresis of

DOX

polyplexesscr-siRNA, where scr-siRNA is negative control and

polymer/scr-siRNA weight ratios are shown above wells with numbers (10-50).


Page 23 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.3. Nanoparticles-blood compatibility 3.3.1. Hemolysis study Hemocompatibility of nanoparticles is generally evaluated by their hemolytic behavior on RBCs. The nanoparticles can interface with RBCs membranes causing their disruption.51 The released hemoglobin from RBCs can be monitored by UV-visible spectrophotometry at a wavelength of 540 nm. Depending on several studies, with a correlation of in vitro and in vivo hemolytic effect of nanocarriers, percent hemolysis ranging from 5-25 % is considered to be “no concern”.52-53 Moreover, in vivo hemolytic behavior of nanocarriers is much lesser than in vitro conditions, due to their interactions with systemic circulation components. The polymeric nanoparticles showed concentration and pH dependent hemolytic behavior (Figure 2). After incubating nanoparticles with RBCs for 1 h, at a highest concentration of 2 mg/mL, ~20 % hemolysis was observed, whereas lower concentration showed lower hemolysis. This minimal heamolytic pattern of developed nanocarriers can be attributed to shielding of excess cationic charges by multiple PEG chains in the polymer. Thus, results showed that developed nanoparticles are hemocompatible over the concentration range of 0.1-2 mg/mL. 3.3.2. Endosomolytic behavior of the nanoparticles The hemolysis assay at low pH is generally used to evaluate the endosomolytic nature or proton sponge behavior of positively charged nanosystems.54-55 After cellular internalization of nanocarriers, they get entrapped in the acidic intracellular organelles such as endosome or lysosome (pH ~ 4.5-6.2). Both drug and siRNA may degrade in such acidic conditions, hence endosomal escape of nanoparticles is necessary through membrane disruption by increasing capacity of “proton sponge effect”. Branched PEI exhibit excellent endosomolytic effect through buffering activity of their secondary and tertiary nitrogen atoms.56-57 This results in an increased influx of protons and counterions into endosomes, increasing osmotic pressure, which leads to swelling and lysis of endosomes and release of nanoparticles in the cytoplasm.58 In this experiment, blank polymeric nanoparticles were incubated in various pH saline buffers, which mimic the endolysosomal pH (pH 5.0) and early endosomal pH (pH 6.2). The hemoglobin detected is directly proportional to buffering capacity of nanoparticles in endolysosomes. The polymeric nanoparticles at a concentration of 2 mg/mL, showed hemolysis of ~ 74 % and ~ 96 % at pH 6.2 and pH 5.0 respectively (Figure 2). The hemolysis was found to be dose dependent at both pH values. In addition, the endosomolytic behavior of blank polymeric nanoparticles prepared by [(HOOC-PEGMA)n-ss-PCL-COOH] copolymer 23 ACS Paragon Plus Environment


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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 43

does not show significant hemolysis in all three pH buffer solutions (Figure S22). This can be attributed to the absence of PEI chains in polymer unit which is responsible for proton sponge effect. Thus the developed nanoformulation has scope for combinatorial delivery of drug and siRNA for better therapeutic efficacy.

Figure 2. Hemocompatibility and proton sponge effect/endosomolytic behavior of the polymeric nanoparticles at endolysosomal pH (pH 5.0), early endosomal pH (pH 6.2), and physiological pH (pH 7.4). (Mean ± SD, n = 3. Where *** indicates p < 0.001 and * indicates p < 0.05).

3.3.3. Coagulation studies After administration, nanoparticles have a tendency to interface with platelets and coagulating co-factors resulting in activation of coagulation pathway. The activated coagulation pathways can result into blockage of blood vessels. Therefore, determination of nanoparticles induced thrombogenicity is a crucial parameter for their hemocompatibility assessment.59 The prothrombin time (PT) is related to the extrinsic pathway, while activated partial prothrombin time (aPTT) is related to the intrinsic coagulation pathway. The significant changes in these 24 ACS Paragon Plus Environment

Page 25 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


two factors is an indication of activation of coagulation cascade by nanoparticles interactions with blood. Polymeric nanoparticles were mixed with blood and incubated for 1 h in order to determine PT and aPTT parameters. Normal values for PT and aPTT for heathy blood is in the range of 11-14 s and 27-40 s respectively.60 The results depict that the PT and aPTT were inside the typical range over the nanoparticles concentration between 0.1-2 mg/mL, which designated that the nanoparticles did not interact with blood coagulation factors (Table 3). The biocompatible nature of nanoformulation towards coagulation system can be attributed to the presence of hydrophilicity bestowed due to PEG and PEI in the outer shell of nanoparticles. Table 3. Coagulation studies of blank polymeric nanoparticlesa. Nanoparticles concentration (mg/mL) Control

PT (s)

aPTT (s)

12.50 ± 0.20

30.90 ± 0.98

0.1

12.33 ± 0.25

33.50 ± 1.06

0.5

12.23 ± 0.67

32.43 ± 0.85

1

12.20 ± 0.44

32.47 ± 0.60

1.5

12.03 ± 0.71

33.20 ± 0.56

2

12.27 ± 0.67

33.67 ± 0.85 a

Mean ± SD, n = 3.

3.4. In vitro drug release In vitro DOX release of DOXnanoparticles was carried out in pH 5 (endosomal pH) and pH 7.4 with and without 10 mmol of GSH (GSH level of tumor cells).61 The release profile was also studied at pH 7.4 with 20 µmol GSH (GSH level of blood and extracellular matrix environment).17, 40 The DOX release was found to be maximum at pH 5 with 10 mmol GSH (~ 60 %), proceeded by pH 7.4 with 10 mmol GSH (~ 44 %) indicating selective DOX release performance of redox sensitive nanoparticles, while the cumulative release profile in acidic pH 5.0 was found to be (~ 36 %) (Figure 3). The DOX release rate from

DOX

nanoparticles in pH

7.4 and with 20 µmol GSH was very slow resulting in cumulative drug release of ~ 14 % and ~ 16 % respectively. This less and steady release profile at physiological pH 7.4, can be credited to stable nature of disulfide linkage in the polymer chain. The 2.4 fold enhanced drug release at pH 5 in comparison to PBS pH 7.4 is credited to the improved hydrophilicity of DOX because of protonation of the glycosidic amine group (DOX pKa is 8.46). Similar type of pH reliant DOX release was also reported in DOX loaded polymeric nanosystems.62-63 Thus, such increased DOX release at pH 5 gives added advantage to nanocarriers for intracellular 25 ACS Paragon Plus Environment


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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 43

augmented drug release within the acidic endosomal/lysosomal compartments. Destabilization of the nanoparticles by cleavage of disulfide bonds of the polymer chains in GSH (10 mmol) could have resulted in further increase in drug release at pH 5 with 10 mmol GSH. Interestingly, the developed nanosystem has GSH triggered drug release property with the added advantage of improved DOX release in low pH.

Figure 3. In vitro drug release profile of optimized DOXnanoparticles formulation at pH 7.4, pH 7.4 with 20 μmol GSH, pH 5, pH 7.4 with 10 mmol GSH and pH 5 with 10 mmol GSH for 72 h. (Mean ± SD, n = 3). 3.5. Colloidal stability of polyplexes Colloidal stability of nanoformulation is an important aspect in clinical point of view. Nanoparticles should uniformly dispersed in aqueous media without showing any aggregation, which helps in their easy cellular penetration. As the aggregation of nanoparticles can cause adverse cell-nanoparticles interactions, it is desirable to study the stability of nanoformulation in different media.64 The colloidal stability of

DOX

polyplexesscr-siRNA was evaluated by

determining changes in hydrodynamic size and PDI using DLS in different media such as PBS pH 7.4 with and without 10 mmol GSH and 20 μmol GSH and DMEM medium supplemented 26 ACS Paragon Plus Environment

Page 27 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with 10 % FBS. The DOXpolyplexesscr-siRNA were found to be stable in PBS pH 7.4 and DMEM medium supplemented with 10 % FBS with size and PDI below 160 nm and 0.35 respectively (Figure 4A, B). Moreover, DOXpolyplexesscr-siRNA were stable in PBS pH 7.4 containing 20 μmol GSH depicting their stability in blood circulation. This stability can be attributed to hydrophilic PEG and PEI chains over the surface of nanoparticles. In addition, PEGylation provides “stealth nature” to nanoparticles giving stearic stabilization in aqueous media and affording long circulation effect to nanoparticles.65 Thus, the stability study demonstrated the excellent extracellular stability and will have fast intracellular drug release due to redox sensitive nature of nanoparticles.

Figure 4. Colloidal stability of

DOX

polyplexesscr-siRNA in PBS pH 7.4, Dulbecco's Modified

Eagle Medium (DMEM) supplemented with 10 % FBS, 20 μmol GSH and 10 mmol GSH for 72 h, determined by Dynamic Laser Scattering (DLS), where (A) Represents size, (B) Represents polydispersity index (PDI). (Mean ± SD, n = 3).



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 43

3.6. Cell culture studies 3.6.1. In vitro cellular uptake and nuclear localization evaluation by using confocal laser scanning microscopy (CLSM)

Cellular uptake study is an essential part for nanosystem, as it can predict the in vivo efficacy of nanoformulation. To check the efficacy of nanoparticles to deliver both DOX and siRNA simultaneously, FAM-siRNA at 100 nmol concentration was used to prepare polyplexes, DOX

polyplexesFAM-siRNA. Excellent co-localization was observed within cytoplasm for both

MDA-MB-231 and HeLa cells after 2 h incubation of DOXpolyplexesFAM-siRNA (Figure 5A, B). The yellow color in the merged image was due to overlapping of fluorescence from DOX and FAM-siRNA. Thus co-localization of DOX and FAM-siRNA depicts the ability of developed nanosystem to deliver both drug and siRNA to cells, concurrently. Further, in both the cell lines nuclear localization of DOX was observed by increasing the incubation time for DOX

polyplexesFAM-siRNA up to 8 h (Figure 5C, D). After increasing incubation time, most of the

drug was found in the nucleus and very small proportion remained in the cytoplasm. 3.6.2. Quantitative evaluation of in vitro cellular uptake by flow cytometry The quantitative assessment of cellular uptake for

DOX

polyplexesFAM-siRNA was evaluated in

both HeLa and MDA-MB-231 cells. The positive fluorescence shift for both DOX and FAMsiRNA was visualised in both cell lines, indicating DOXpolyplexesFAM-siRNA indeed was able to deliver both payloads simultaneously (Figure 5E1, E2). The results of flow cytometry for concurrent delivery of DOX and FAM-siRNA corroborates the observations obtained in confocal cellular uptake studies.


Page 29 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. CLSM images depicting the simultaneous delivery of DOX and FAM-siRNA through DOX

polyplexesFAM-siRNA, in (A) MDA-MB-231 and (B) HeLa cells after 2 h incubation. CLSM

images for nuclear localization of DOX by DOXpolyplexesFAM-siRNA in (C) MDA-MB-231 and (D) HeLa cells after 8 h incubation. Images were taken at 60 X magnification. Subtitle figures, (E1) and (E2) Represents the quantitative cellular uptake by FACS for FAM-siRNA and DOX in MDA-MB-231 and HeLa cells delivered through

DOX

polyplexesFAM-siRNA after 2 h

incubation.



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 43

3.6.3. Real time PCR analysis Polo like kinase 1 (plk1), an endogenous enzyme which is overexpressed in tumor cells and correlates with aggressive behavior is often chosen as target tumor cell growth inhibition.10, 66 Moreover, the low inhibitory effect of plk1 siRNA on normal cells than tumor cells makes plk1 an interesting candidate for cancer therapy.41, 67 Polo like kinases play a central role in mitotic checkpoints and regulates cell cycle process through G2 and M phase. They help in formation and changes in spindle formation, hence overexpression of this kinase can lead to uncontrolled cell division with aneuploidy, which ultimately leads to cancer.68 Herein, we have verified polyplexes efficiency to knockdown the expression of therapeutic target gene, plk1 by RT-PCR. Polyplexesplk1-siRNA and polyplexesscr-siRNA were incubated with MDA-MB-231 and HeLa cells for 48 h and then plk1 mRNA expression was detected using real-time PCR. The plk1 mRNA expression was reduced in dose dependent manner when treated with polyplexesplk1-siRNA. For example, 50, 100 and 125 nmol of plk1 lead to approximately 45%, 60% and 70% knockdown of plk1 mRNA in HeLa cells, respectively, whereas knockdown efficiency in MDA-MB-231 cells was found to be 50 %, 65 % and 70 % respectively (Figure 6A). Cells treated with polyplexesscr-siRNA at 50, 100 and 125 nmol concentrations did not show any significant knockdown and exhibited almost same gene expression as that of untreated cells. 3.6.4. In vitro toxicity assessment of nanoparticles The cytocompatibility of blank polymeric nanoparticles was evaluated by MTT assay in MDAMB-231 and HeLa cells over the concentration range of 0.1-2 mg/mL, no significant cytotoxicity was overserved in both cell lines, depicting its cytocompatible nature (Figure 6B). Moreover, the effect of ROS generation by polymeric nanoparticles was also studied, where nanoparticles did not show any significant increase in ROS production (Supporting information, S3 and Figure S23). Thus developed nanocarriers did not possess any potential to increase intracellular free radicals. The non-cytotoxicity of polymeric nanoparticles indicates their biosafety, which can be further safely employed for biological applications.

The

synergistic effect of DOX and plk1 siRNA through DOXpolyplexesplk1-siRNA was assessed using MTT assay in aforementioned cell lines, where DOXpolyplexesscr-siRNA formulation was used for comparison. The dose dependent cytotoxicity was observed in both cell lines, where DOX

polyplexesplk1-siRNA showed significantly more toxicity than

6C, D). The IC50 values for

DOX

polyplexesscr-siRNA (Figure

DOX

polyplexesplk1-siRNA in MDA-MB-231 and HeLa cells were 30

ACS Paragon Plus Environment

Page 31 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


found to 1.77 μg/mL and 2.55 μg/mL, while for

DOX

polyplexesscr-siRNA IC50 values were 2.25

μg/mL and 5.11 μg/mL respectively. This low IC50 value for

DOX

polyplexesplk1-siRNA is

attributed to synergistic effect of both DOX and plk1-siRNA.

Figure 6. (A) The percent plk1 mRNA expression in MDA-MB-231 and HeLa cells transfected with polyplexesplk1-siRNA and polyplexesscr-siRNA (polymer/siRNA weight ratio 15). The percent plk1 mRNA expression is represented with respect to control (untreated cells). Figure (B) Represents cell viability assay for blank polymeric nanoparticles. Figure (C) and (D) Represents effect of cell proliferation of

DOX

polyplexesscr-siRNA and

DOX

polyplexesplk1-siRNA on

MDA-MB-231 and HeLa cells respectively, where siRNA concentration was kept constant (100 nmol). (Mean ± SD, n = 3. Where *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05, “ns” indicates not significant, p > 0.05). 3.7. Animal studies 3.7.1. Anti tumor efficacy in EAT tumor model The synergistic effect of the dual cargos loaded cationic nanoparticles was further evaluated in vivo using EAT tumor model. The control group administered with PBS pH 7.4 showed rapid tumor growth compared to all other treatment groups (Figure 7A, A1). The administration of polyplexesplk1-siRNA caused 3.45 fold reduction in percent relative tumor volume, compared to 31 ACS Paragon Plus Environment


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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

control.

Page 32 of 43

DOX

polyplexesscr-siRNA and free DOX treated groups shown 14.02 and 16.62 fold

decrease in tumor volume with respect to control group respectively. More interestingly, codelivery of DOX and plk1 siRNA through DOXpolyplexesplk1-siRNA formulation depicted a most significant reduction in the percent relative tumor volume (29.01 fold decrease, p-value < 0.001). A Kaplan-Meier survival plot (Figure 7B) showed that life span of mice treated with DOX

polyplexesplk1-siRNA was increased significantly. The median survival time (MST) for

DOX

polyplexesplk1-siRNA,

DOX

polyplexesscr-siRNA, DOX, polyplexesplk1-siRNA and control groups

was found to be 46, 41, 39, 36 and 27 days respectively. The body weight of control group was found to be significantly increasing than polyplexes treated groups during the indicated experimental period (p-value < 0.001). This can be attributed to increasing tumor volume in control group, during the course of the experiment. While no significant difference was observed in body weights of polyplexes treated groups (Figure S24B). Thus, in vivo tumor regression results indicate the efficacy of DOXpolyplexesplk1-siRNA for the synergistic antitumor effect of loaded moieties in tumor cells. Excitingly, in DOXpolyplexesplk1-siRNA treated group, the tumor was not visible in three out of six mice. The images of excised tumor at the end point of study are depicted in figure 7C.

Figure 7. Percent tumor growth curves for antitumor effect after intratumoral administration of PBS pH 7.4 (Control), Polyplexesplk1-siRNA, DOX, siRNA.

DOX

polyplexesscr-siRNA,

DOX

polyplexesplk1-

(A and A1), where DOX and plk1-siRNA were administered at a cumulative dose of 6

mg/kg and 2 mg/kg of body weight respectively. (Mean ± SEM, n = 6. Where *** indicates p < 0.001). (B) Kaplan-Meier survival plot of tumor containing mice, dotted lines represent dose 32 ACS Paragon Plus Environment

Page 33 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


administration time points. (C) Tumor images of mice at the end point of study treated with different formulations, (i) PBS pH 7.4 (control), (ii) Polyplexesplk1-siRNA, (iii) DOX, (iv) DOX

polyplexesscr-siRNA, (v) DOXpolyplexesplk1-siRNA.

3.7.2. Toxicity evaluation of the developed nanosystems in vivo with serum biochemical parameters and histopathological evaluation of vital organs To evaluate the safety and biocompatibility of developed cationic nanoparticles in vivo, eleven days repeated dose toxicity study was performed in female Swiss Albino mice. The dose (100 mg/kg) of blank polymeric nanoparticles was selected based on previously reported studies.42, 69

The cationic nanoparticles treated group did not show any significant change in body weights

compared to the control group (Figure S24A). Serum biochemistry and hematological parameters were comparable between control and treated groups (Table S2). Histopathological evaluation of the vital organs of nanoparticles treated group did not show any toxicity caused by the accumulation of nanoparticles (Figure S25). Thus, the data suggest that the developed nanocarriers are safe for in vivo drug delivery applications. The major serum biochemical parameters of all groups of the tumor inhibition study were studied. No significant difference was observed in nanoparticles treated groups and control (Table 4). Also, DOX treated did not show any significant alteration in serum biochemistry parameters. This is attributed to the chosen route of administration as intratumoral and also the administration of low cumulative DOX dose (6 mg/kg).



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 43

Table 4. Serum biochemical parameters of animals treated with different nanoformulations used in tumor regression studya. Serum Dox

polyplexesscr-

biochemistry

Control

Dox

polyplexesplk1

Polyplexesplk1-

Dox sirna

-sirna

sirna

parametersb CK-MB (IU/L)

2.71 ± 0.25

2.95 ± 0.16

2.63 ± 0.75

2.31 ± 0.81

2.38 ± 0.60

AST (IU/L)

66.00 ± 4.58

74.00 ± 7.55

67.33 ± 4.73

71.00 ± 8.19

74.67 ± 7.57

ALT (IU/L)

51.33 ± 3.51

54.00 ± 6.56

48.67 ± 8.02

68.67 ± 9.02

56.00 ± 9.17

Urea (mg/dl)

41.67 ± 5.51

38.00 ± 2.00

39.33 ± 8.62

41.33 ± 7.02

45.67 ± 5.03

Creatinine (mg/dl)

0.28 ± 0.03

0.28 ± 0.07

0.31 ± 0.01

0.27 ± 0.10

0.26 ± 0.06

(aMean ± SD, n = 3) b

Where, CK-MB: Creatine Kinase-MB, AST: Alanine aminotransferase and ALT: Aspartate

aminotransferase. Histopathological evaluation of the vital organs did not show any significant toxicity in all nanoparticles treated groups (Figure 8A-E). Control group tumor sections displayed more viable cells than that of nanoparticles treated groups, where purple color shows viable cells while light pink color indicates necrotic area. The

DOX

polyplexesplk1-siRNA treated group

exhibited maximum tumor necrosis than all other groups. In a microscopic examination of the heart, hyalinized blood vessels and bundles of cardiac muscle fibers were observed without any significant toxicity caused by DOX. Lung sections were analyzed by bronchial epithelium which was lined by stratified ciliated columnar epithelium. Additionally, maximum bronchi and bronchioles walls were visualized with smooth muscle fibers. Lung alveolar space in all treated groups was found to be empty with expanded septa and filled with a mononuclear cell infiltrate and polymorphs without any granuloma. In liver sections, uniform size polygonal cells in all three zones were seen in all groups, while only mild ballooning degeneration was seen in the eosinophilic cytoplasm. No pigment deposition, fibrosis and steatosis were observed in all groups. In the case of kidney evaluation, the presence of healthy glomeruli, interstitium, tubules and blood vessels were detected in all groups. Microscopic evaluation of spleen disclosed normal myelopoiesis, nucleated erythroblasts and lymphoid tissue. Thus, as all the microscopic observations for nanoparticles treated groups were more or less similar to control group, indicating no toxicity to all vital organ from any polyplexes formulation. 34 ACS Paragon Plus Environment

Page 35 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Histopathological evaluation of vital tumors and vital organs of mice groups employed in tumor regression study, where (A) Control, PBS pH 7.4 treated, (B) Polyplexesplk1siRNA

treated, (C) DOX treated, (D)

DOX

polyplexesplk1-siRNA treated, (E)

DOX

polyplexesscr-siRNA

treated. All images were taken at 20 X magnification.

4. CONCLUSIONS We have demonstrated simultaneous delivery of a chemotherapeutic drug and siRNA using a biocompatible, biodegradable and redox sensitive cationic polymeric nanosystem, in vitro and in vivo. The “two-in-one” drug loaded polyplexes cocktail with redox sensitive property depicted significantly improved clinical efficacy, in comparison to formulations loaded with a single active agent. Drug release from the polyplexes could be regulated by low pH and redox conditions, suggesting their ability for tumor specific delivery. The fascinating and promising features of this nanosystem, in comparison to existing systems, are the utilization of short polyethylene glycol chains which has a negligible chance of evoking any immune response and low molecular weight PEI which is safer to the cells. Further, multiple short PEG chains can provide “stealth” nature to nanosystem thereby providing excellent stability for developed nanoformulation in physiological media. Self-assembly of the developed nanocarriers indicate their feasibility for industrial scale up. Polyplexes were able to downregulate the plk1 gene expression and depicted remarkable tendency to inhibit tumor growth in a synergistic manner. 35 ACS Paragon Plus Environment


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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 43

Considering all the aforementioned aspects, the developed nanosystem is a potential candidate to be transformed into preclinical and clinical settings.

ASSOCIATED CONTENTS Supporting information Determination of primary amine content of the nanoparticles, protein adsorption study, evaluation of the nanoparticles for redox oxygen species (ROS) generation, polymer characterization data, including 1H NMR, HRMS,

13

C NMR, GPC, FTIR, CHNS analysis.

Dynamic Laser Scattering studies for blank and DOX loaded nanoparticles, PXRD study for assessment of DOX characteristic in nanoparticles, relative body weight changes of animals employed for toxicity evaluation study and tumor regression study, Histopathological evaluation of vital organs of mice used for toxicity evaluation study. Hematology and serum biochemistry parameters analysis for mice employed for toxicity evaluation study.

AUTHOR INFORMATION Corresponding Author *Tel.: +91 11 26591041. E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS Authors are grateful to the Department of Biotechnology (DBT), India, for the research grant (BT/PR10191/NNT/28/717/2013). We gratefully acknowledge to Dr. Immanual from department of pathology and Mrs. Archana Bansal from the department of cardiology, All India Institute of Medical Sciences, New Delhi, India, for their help in histopathology and serum biochemistry analysis. We acknowledge Mr. Anil Pandey for his help in animal studies. We sincerely acknowledge Nanoscale Research Facility, IIT Delhi, India for AFM study. Mr. Chetan Nehate is also thankful to the Indian Institute of Technology, New Delhi, India for granting him a research assistantship.


Page 37 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A. Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis Elegans. Nature. 1998, 391 (6669), 806-811. (2) Xu, X.; Wu, J.; Liu, Y.; Saw, P. E.; Tao, W.; Yu, M.; Zope, H.; Si, M.; Victorious, A.; Rasmussen, J. Multifunctional Envelope-Type siRNA Delivery Nanoparticle Platform for Prostate Cancer Therapy. ACS nano. 2017, 11 (3), 2618-2627. (3) Zheng, X.; Pang, X.; Yang, P.; Wan, X.; Wei, Y.; Guo, Q.; Zhang, Q.; Jiang, X. A Hybrid siRNA Delivery Complex for Enhanced Brain Penetration and Precise Amyloid Plaque Targeting in Alzheimer’s Disease Mice. Acta Biomater. 2017, 49, 388-401. (4) Wang, T.; Shigdar, S.; Al Shamaileh, H.; Gantier, M. P.; Yin, W.; Xiang, D.; Wang, L.; Zhou, S.-F.; Hou, Y.; Wang, P. Challenges and Opportunities for siRNA-Based Cancer Treatment. Cancer Lett. 2017, 387, 77-83. (5) Tatiparti, K.; Sau, S.; Kashaw, S. K.; Iyer, A. K. siRNA Delivery Strategies: A Comprehensive Review of Recent Developments. Nanomaterials. 2017, 7 (4), 77. (6) Pharmaceuticals, C. T., Study to Evaluate the Safety, Tolerability, Pharmacokinetics (PK), and Pharmacodynamics (PD), of Liposomal siRNA in Subjects with High Cholesterol. Available from Url: https://clinicaltrials.gov/ct2/show/NCT00927459. 2010 (7) Amjad, M. W.; Kesharwani, P.; Amin, M. C. I. M.; Iyer, A. K. Recent Advances in the Design, Development, and Targeting Mechanisms of Polymeric Micelles for Delivery of siRNA in Cancer Therapy. Prog. Polym. Sci. 2017, 64, 154-181. (8) Sarisozen, C.; Pan, J.; Dutta, I.; Torchilin, V. P. Polymers in the Co-delivery of siRNA and Anticancer Drugs to Treat Multidrug-Resistant Tumors. J. Pharm. Invest. 2017, 1-13. (9) Xiong, X.-B.; Lavasanifar, A. Traceable Multifunctional Micellar Nanocarriers for CancerTargeted Co-delivery of MDR-1 siRNA and Doxorubicin. ACS nano. 2011, 5 (6), 5202-5213. (10) Sun, T.-M.; Du, J.-Z.; Yao, Y.-D.; Mao, C.-Q.; Dou, S.; Huang, S.-Y.; Zhang, P.-Z.; Leong, K. W.; Song, E.-W.; Wang, J. Simultaneous Delivery of siRNA and Paclitaxel via a “Two-in-One” Micelleplex Promotes Synergistic Tumor Suppression. ACS nano. 2011, 5 (2), 1483-1494. (11) Kim, D.; Gao, Z. G.; Lee, E. S.; Bae, Y. H. In Vivo Evaluation of Doxorubicin-Loaded Polymeric Micelles Targeting Folate Receptors and Early Endosomal pH in Drug-Resistant Ovarian Cancer. Mol. Pharmaceutics. 2009, 6 (5), 1353-1362. (12) Xu, C.; Wang, P.; Zhang, J.; Tian, H.; Park, K.; Chen, X. Pulmonary Codelivery of Doxorubicin and siRNA by pH‐Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer. Small. 2015, 11 (34), 4321-4333.



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 43

(13) Ebrahimian, M.; Taghavi, S.; Mokhtarzadeh, A.; Ramezani, M.; Hashemi, M. Co-delivery of Doxorubicin Encapsulated PLGA Nanoparticles and Bcl-xL shRNA Using Alkyl-Modified PEI into Breast Cancer Cells. Appl. Biochem. Biotechnol. 2017, 1-11. (14) Deng, Z. J.; Morton, S. W.; Ben-Akiva, E.; Dreaden, E. C.; Shopsowitz, K. E.; Hammond, P. T. Layer-by-Layer Nanoparticles for Systemic Codelivery of an Anticancer Drug and siRNA for Potential Triple-Negative Breast Cancer Treatment. ACS nano. 2013, 7 (11), 9571-9584. (15) Quinn, J. F.; Whittaker, M. R.; Davis, T. P. Glutathione Responsive Polymers and Their Application in Drug Delivery Systems. Polym. Chem. 2017, 8 (1), 97-126. (16) Wu, J.; Zhang, J.; Deng, C.; Meng, F.; Cheng, R.; Zhong, Z. Robust, Responsive, and Targeted PLGA Anticancer Nanomedicines by Combination of Reductively Cleavable Surfactant and Covalent Hyaluronic Acid Coating. ACS Appl. Mater. Interfaces. 2017, 9 (4), 3985-3994. (17) Cheng, R.; Feng, F.; Meng, F.; Deng, C.; Feijen, J.; Zhong, Z. Glutathione-Responsive Nano-Vehicles as a Promising Platform for Targeted Intracellular Drug and Gene Delivery. J. Controlled Release. 2011, 152 (1), 2-12. (18) Kuppusamy, P.; Li, H.; Ilangovan, G.; Cardounel, A. J.; Zweier, J. L.; Yamada, K.; Krishna, M. C.; Mitchell, J. B. Noninvasive Imaging of Tumor Redox Status and Its Modification by Tissue Glutathione Levels. Cancer Res. 2002, 62 (1), 307-312. (19) Zhai, S.; Hu, X.; Hu, Y.; Wu, B.; Xing, D. Visible Light-Induced Crosslinking and Physiological Stabilization of Diselenide-Rich Nanoparticles for Redox-Responsive Drug Release and Combination Chemotherapy. Biomaterials. 2017, 121, 41-54. (20) Zhang, F.; Gong, S.; Wu, J.; Li, H.; Oupicky, D.; Sun, M. CXCR4-Targeted and Redox Responsive Dextrin Nanogel for Metastatic Breast Cancer Therapy. Biomacromolecules. 2017, 18 (6), 1793-1802. (21) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12 (11), 991-1003 (22) Lale, S. V.; Kumar, A.; Prasad, S.; Bharti, A. C.; Koul, V. Folic Acid and Trastuzumab Functionalized Redox Responsive Polymersomes for Intracellular Doxorubicin Delivery in Breast Cancer. Biomacromolecules. 2015, 16 (6), 1736-1752. (23) Kumar, A.; Lale, S. V.; Mahajan, S.; Choudhary, V.; Koul, V. Rop and Atrp Fabricated Dual Targeted Redox Sensitive Polymersomes Based on pPEGMA-PCL-ss-PCL-pPEGMA Triblock Copolymers for Breast Cancer Therapeutics. ACS Appl. Mater. Interfaces. 2015, 7 (17), 9211-9227. (24) Kumar, A.; Lale, S. V.; Alex, M. A.; Choudhary, V.; Koul, V. Folic Acid and Trastuzumab Conjugated Redox Responsive Random Multiblock Copolymeric Nanocarriers for Breast Cancer Therapy: In-Vitro and in-Vivo Studies. Colloids Surf., B. 2017, 149, 369-378. (25) Nehate, C.; Alex, M. A.; Kumar, A.; Koul, V. Combinatorial Delivery of Superparamagnetic Iron Oxide Nanoparticles (γFe2O3) and Doxorubicin Using Folate 38 ACS Paragon Plus Environment

Page 39 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Conjugated Redox Sensitive Multiblock Polymeric Nanocarriers for Enhancing the Chemotherapeutic Efficacy in Cancer Cells. Mater. Sci. Eng. C. 2017, 75, 1128-1143. (26) Alex, M. A.; Nehate, C.; Veeranarayanan, S.; Kumar, D. S.; Kulshreshtha, R.; Koul, V. Self Assembled Dual Responsive Micelles Stabilized with Protein for Co-Delivery of Drug and siRNA in Cancer Therapy. Biomaterials. 2017, 133, 94-106. (27) Judge, A. D.; Robbins, M.; Tavakoli, I.; Levi, J.; Hu, L.; Fronda, A.; Ambegia, E.; McClintock, K.; MacLachlan, I. Confirming the RNAi-mediated Mechanism of Action of siRNA-based Cancer Therapeutics in Mice. J. Clin. Investig. 2009, 119 (3), 661-673. (28) Barata, P.; Sood, A. K.; Hong, D. S. RNA-Targeted Therapeutics in Cancer Clinical Trials: Current Status and Future Directions. Cancer Treat. Rev. 2016, 50, 35-47. (29) Yap, T. A.; Carden, C. P.; Kaye, S. B. Beyond Chemotherapy: Targeted Therapies in Ovarian Cancer. Nature reviews. Cancer. 2009, 9 (3), 167-181. (30) Moebus, V.; Jackisch, C.; Lueck, H.-J.; du Bois, A.; Thomssen, C.; Kurbacher, C.; Kuhn, W.; Nitz, U.; Schneeweiss, A.; Huober, J. Intense Dose-dense Sequential Chemotherapy with Epirubicin, Paclitaxel, and Cyclophosphamide Compared with Conventionally Scheduled Chemotherapy in High-Risk Primary Breast Cancer: Mature Results of an Ago Phase Iii Study. J. Clin. Oncol. 2010, 28 (17), 2874-2880. (31) Schöttler, S.; Becker, G.; Winzen, S.; Steinbach, T.; Mohr, K.; Landfester, K.; Mailänder, V.; Wurm, F. R. Protein Adsorption Is Required for Stealth Effect of Poly(Ethylene Glycol)and Poly(Phosphoester)-coated Nanocarriers. Nat. Nanotechnol. 2016, 11 (4), 372-377. (32) Khansarizadeh, M.; Mokhtarzadeh, A.; Rashedinia, M.; Taghdisi, S.; Lari, P.; Abnous, K.; Ramezani, M. Identification of Possible Cytotoxicity Mechanism of Polyethylenimine by Proteomics Analysis. Hum. Exp. Toxicol. 2016, 35 (4), 377-387. (33) Gwak, S.-J.; Nice, J.; Zhang, J.; Green, B.; Macks, C.; Bae, S.; Webb, K.; Lee, J. S. Cationic, Amphiphilic Copolymer Micelles as Nucleic Acid Carriers for Enhanced Transfection in Rat Spinal Cord. Acta Biomater. 2016, 35, 98-108. (34) Liu, S.; Zhou, D.; Yang, J.; Zhou, H.; Chen, J.; Guo, T. Bioreducible Zinc (Ii)Coordinative Polyethylenimine with Low Molecular Weight for Robust Gene Delivery of Primary and Stem Cells. J. Am. Chem. Soc. 2017, 139 (14), 5102-5109. (35) Zheng, M.; Zhong, Z.; Zhou, L.; Meng, F.; Peng, R.; Zhong, Z. Poly (Ethylene Oxide) Grafted with Short Polyethylenimine Gives DNA Polyplexes with Superior Colloidal Stability, Low Cytotoxicity, and Potent in Vitro Gene Transfection under Serum Conditions. Biomacromolecules. 2012, 13 (3), 881-888. (36) Evans, B. C.; Nelson, C. E.; Shann, S. Y.; Beavers, K. R.; Kim, A. J.; Li, H.; Nelson, H. M.; Giorgio, T. D.; Duvall, C. L. Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of PH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs. J. Visualized Exp. 2013, (73). e50166



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 43

(37) Lale, S. V.; Kumar, A.; Naz, F.; Bharti, A. C.; Koul, V. Multifunctional ATRP Based pH Responsive Polymeric Nanoparticles for Improved Doxorubicin Chemotherapy in Breast Cancer by Proton Sponge Effect/Endo-Lysosomal Escape. Polym. Chem. 2015, 6 (11), 21152132. (38) Yildirim, A.; Ozgur, E.; Bayindir, M. Impact of Mesoporous Silica Nanoparticle Surface Functionality on Hemolytic Activity, Thrombogenicity and Non-Specific Protein Adsorption. J. Mater. Chem. B. 2013, 1 (14), 1909-1920. (39) Cheng, R.; Meng, F.; Deng, C.; Klok, H.-A.; Zhong, Z. Dual and Multi-Stimuli Responsive Polymeric Nanoparticles for Programmed Site-Specific Drug Delivery. Biomaterials. 2013, 34 (14), 3647-3657. (40) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12 (11), 991-1003. (41) Alex, M. A.; Veeranarayanan, S.; Poulose, A. C.; Nehate, C.; Kumar, D. S.; Koul, V. Click Modified Amphiphilic Graft Copolymeric Micelles of Poly (Styrene-Alt-Maleic Anhydride) for Combinatorial Delivery of Doxorubicin and Plk-1 siRNA in Cancer Therapy. J. Mater. Chem. B. 2016, 4 (45), 7303-7313. (42) Titlow, W. B.; Lee, C.-H.; Ryou, C. Characterization of Toxicological Properties of LLysine Polymers in CD-1 Mice. J. Microbiol. Biotechnol. 2013, 23 (7), 1015-1022. (43) Datir, S. R.; Das, M.; Singh, R. P.; Jain, S. Hyaluronate Tethered,“Smart” Multiwalled Carbon Nanotubes for Tumor-Targeted Delivery of Doxorubicin. Bioconjugate Chem. 2012, 23 (11), 2201-2213. (44) Zhong, X.; Yang, K.; Dong, Z.; Yi, X.; Wang, Y.; Ge, C.; Zhao, Y.; Liu, Z. Polydopamine as a Biocompatible Multifunctional Nanocarrier for Combined Radioisotope Therapy and Chemotherapy of Cancer. Adv. Funct. Mater. 2015, 25 (47), 7327-7336. (45) Florea, B. I.; Meaney, C.; Junginger, H. E.; Borchard, G. Transfection Efficiency and Toxicity of Polyethylenimine in Differentiated Calu-3 and Nondifferentiated COS-1 Cell Cultures. AAPS J. 2002, 4 (3), 1-11. (46) Guo, Z.; Chen, J.; Lin, L.; Guan, X.; Sun, P.; Chen, M.; Tian, H.; Chen, X. pH Triggered Size Increasing Gene Carrier for Efficient Tumor Accumulation and Excellent Anti-Tumor Effect. ACS Appl. Mater. Interfaces. 2017, 9 (18), 15297-15306. (47) Pishavar, E.; Shafiei, M.; Mehri, S.; Ramezani, M.; Abnous, K. The Effects of Polyethylenimine/DNA Nanoparticle on Transcript Levels of Apoptosis-Related Genes. Drug Chem. Toxicol. 2017, 1-4. (48) Fessi, H.; Puisieux, F.; Devissaguet, J. P.; Ammoury, N.; Benita, S. Nanocapsule Formation by Interfacial Polymer Deposition Following Solvent Displacement. Int. J. Pharm. 1989, 55 (1), R1-R4. (49) Deng, H.; Liu, J.; Zhao, X.; Zhang, Y.; Liu, J.; Xu, S.; Deng, L.; Dong, A.; Zhang, J. PEGb-PCL Copolymer Micelles with the Ability of pH-Controlled Negative-to-Positive Charge 40 ACS Paragon Plus Environment

Page 41 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Reversal for Intracellular Delivery of Doxorubicin. Biomacromolecules. 2014, 15 (11), 42814292. (50) Wu, J.; Song, C.; Jiang, C.; Shen, X.; Qiao, Q.; Hu, Y. Nucleolin Targeting As1411 Modified Protein Nanoparticle for Antitumor Drugs Delivery. Mol. Pharmaceutics. 2013, 10 (10), 3555-3563. (51) Glass, J. J.; Chen, L.; Alcantara, S.; Crampin, E. J.; Thurecht, K. J.; De Rose, R.; Kent, S. J. Charge Has a Marked Influence on Hyperbranched Polymer Nanoparticle Association in Whole Human Blood. ACS Macro Lett. 2017, 6, 586-592. (52) Krzyzaniak, J. F.; Nuúnñez, F. A. A.; Raymond, D. M.; Yalkowsky, S. H. Lysis of Human Red Blood Cells. 4. Comparison of in Vitro and in Vivo Hemolysis Data. J. Pharm. Sci. 1997, 86 (11), 1215-1217. (53) Fort, F. L.; Heyman, I. A.; Kesterson, J. W. Hemolysis Study of Aqueous Polyethylene Glycol 400, Propylene Glycol and Ethanol Combinations in Vivo and in Vitro. PDA J. Pharm. Sci. Technol. 1984, 38 (2), 82-87. (54) Xiao, K.; Li, Y.; Luo, J.; Lee, J. S.; Xiao, W.; Gonik, A. M.; Agarwal, R. G.; Lam, K. S. The Effect of Surface Charge on in Vivo Biodistribution of Peg-Oligocholic Acid Based Micellar Nanoparticles. Biomaterials. 2011, 32 (13), 3435-3446. (55) Henry, S. M.; El-Sayed, M. E.; Pirie, C. M.; Hoffman, A. S.; Stayton, P. S. pH-Responsive Poly (Styrene-Alt-Maleic Anhydride) Alkylamide Copolymers for Intracellular Drug Delivery. Biomacromolecules. 2006, 7 (8), 2407-2414. (56) Guo, S.; Huang, Y.; Jiang, Q.; Sun, Y.; Deng, L.; Liang, Z.; Du, Q.; Xing, J.; Zhao, Y.; Wang, P. C. Enhanced Gene Delivery and siRNA Silencing by Gold Nanoparticles Coated with Charge-Reversal Polyelectrolyte. ACS nano. 2010, 4 (9), 5505-5511. (57) Tu, J.; Wang, T.; Shi, W.; Wu, G.; Tian, X.; Wang, Y.; Ge, D.; Ren, L. Multifunctional Znpc-Loaded Mesoporous Silica Nanoparticles for Enhancement of Photodynamic Therapy Efficacy by Endolysosomal Escape. Biomaterials. 2012, 33 (31), 7903-7914. (58) Seo, K.; Kim, D. Ph-Dependent Hemolysis of Biocompatible Imidazole-Grafted Polyaspartamide Derivatives. Acta Biomater. 2010, 6 (6), 2157-2164. (59) Ilinskaya, A. N.; Dobrovolskaia, M. A. Nanoparticles and the Blood Coagulation System. Part II: Safety Concerns. Nanomedicine. 2013, 8 (6), 969-981. (60) Santoro, S.; Eby, C. Laboratory Evaluation of Hemostatic Disorders. Hematology. Basic principles and practice. 3rd ed. New York: Churchill Livingstone 2000, 1841-50. (61) Zhao, Q.; Geng, H.; Wang, Y.; Gao, Y.; Huang, J.; Wang, Y.; Zhang, J.; Wang, S. Hyaluronic Acid Oligosaccharide Modified Redox-Responsive Mesoporous Silica Nanoparticles for Targeted Drug Delivery. ACS Appl. Mater. Interfaces. 2014, 6 (22), 2029020299.



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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 43

(62) Kataoka, K.; Matsumoto, T.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Fukushima, S.; Okamoto, K.; Kwon, G. S. Doxorubicin-Loaded Poly(Ethylene Glycol)-Poly(β-Benzyl-LAspartate) Copolymer Micelles: Their Pharmaceutical Characteristics and Biological Significance. J. of Controlled Release. 2000, 64 (1), 143-153. (63) Cao, N.; Cheng, D.; Zou, S.; Ai, H.; Gao, J.; Shuai, X. The Synergistic Effect of Hierarchical Assemblies of siRNA and Chemotherapeutic Drugs Co-delivered into Hepatic Cancer Cells. Biomaterials. 2011, 32 (8), 2222-2232. (64) Sharma, R.; Lee, J.-S.; Bettencourt, R. C.; Xiao, C.; Konieczny, S. F.; Won, Y.-Y. Effects of the Incorporation of a Hydrophobic Middle Block into a PEG-Polycation Diblock Copolymer on the Physicochemical and Cell Interaction Properties of the Polymer-DNA Complexes. Biomacromolecules. 2008, 9 (11), 3294-3307. (65) Yang, C.; Gao, S.; Dagnæs-Hansen, F.; Jakobsen, M.; Kjems, J. Impact of PEG Chain Length on the Physical Properties and Bioactivity of PEGylated Chitosan/siRNA Nanoparticles in Vitro and in Vivo. ACS Appl. Mater. Interfaces. 2017, 9 (14), 12203-12216. (66) Lin, D.; Cheng, Q.; Jiang, Q.; Huang, Y.; Yang, Z.; Han, S.; Zhao, Y.; Guo, S.; Liang, Z.; Dong, A. Intracellular Cleavable Poly (2-Dimethylaminoethyl Methacrylate) Functionalized Mesoporous Silica Nanoparticles for Efficient siRNA Delivery in Vitro and in Vivo. Nanoscale. 2013, 5 (10), 4291-4301. (67) Xu, C.-F.; Zhang, H.-B.; Sun, C.-Y.; Liu, Y.; Shen, S.; Yang, X.-Z.; Zhu, Y.-H.; Wang, J. Tumor Acidity-Sensitive Linkage-Bridged Block Copolymer for Therapeutic siRNA Delivery. Biomaterials. 2016, 88, 48-59. (68) Lee, S.-Y.; Jang, C.; Lee, K.-A. Polo-Like Kinases (Plks), a Key Regulator of Cell Cycle and New Potential Target for Cancer Therapy. Dev Reprod. 2014, 18 (1), 65-71. (69) Knudsen, K. B.; Northeved, H.; Ek, P. K.; Permin, A.; Gjetting, T.; Andresen, T. L.; Larsen, S.; Wegener, K. M.; Lykkesfeldt, J.; Jantzen, K. In Vivo Toxicity of Cationic Micelles and Liposomes. Nanomedicine. 2015, 11 (2), 467-477.


Page 43 of 43

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of contents (TOC):


ATRP Fabricated and Short Chain Polyethylenimine Grafted Redox

Recommend Documents