Chitosan grafted-poly(ethylene glycol) methacrylate nanoparticles as carrier for controlled release of bevacizumab

Accepted Manuscript Chitosan grafted-poly(ethylene glycol) methacrylate nanoparticles as carrier for controlled release of bevacizumab

Corina-Lenuța Savin, Marcel Popa, Christelle Delaite, Marcel Costuleanu, Danut Costin, Catalina Anișoara Peptu PII: DOI: Reference:

S0928-4931(18)32263-X https://doi.org/10.1016/j.msec.2019.01.036 MSC 9294

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

31 July 2018 4 December 2018 8 January 2019

Please cite this article as: Corina-Lenuța Savin, Marcel Popa, Christelle Delaite, Marcel Costuleanu, Danut Costin, Catalina Anișoara Peptu , Chitosan grafted-poly(ethylene glycol) methacrylate nanoparticles as carrier for controlled release of bevacizumab. Msc (2019), https://doi.org/10.1016/j.msec.2019.01.036

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Chitosan grafted

–

poly(ethylene glycol) methacrylate

nanoparticles as carrier for controlled release of bevacizumab Authors: Corina-Lenuța Savina,b, Marcel Popaa,d, Christelle Delaiteb, Marcel Costuleanuc,

Department of Natural and Synthetic Polymers, Faculty of Chemical Engineering and

RI

a

PT

Danut Costinc, Catalina Anișoara Peptua

Environmental Protection, “Gheorghe Asachi” Technical University of Iaşi, Romania Laboratory of Photochemistry and Macromolecular Engineering Institute J.B. Donnet,

University of Haute Alsace, Mulhouse, France

SC

b

“Grigore T. Popa” University of Medicine and Pharmacy, University Str., Iaşi 16, Romania

d

Academy of Romanian Scientists, Bucuresti, Romania

MA

NU

c

ABSTRACT

D

The aim of the present study is to obtain, for the first time, polymeric nanocarriers based on

PT E

the chitosan grafted-poly(ethylene glycol) methacrylate derivative. The strategy involves the use of chitosan grafted-poly(ethylene glycol) methacrylate with high solubility in water, obtained via Michael addition, in order to prepare potentially non-toxic micro/nanoparticles

CE

(MNPs). By modifying chitosan, its solubility in aqueous media was improved. Micro/nanoparticles-based chitosan grafted-poly(ethylene glycol) methacrylate were obtained under mild condition, with good and controlled swelling properties in acetate buffer solution

AC

(ABS) and phosphate buffer solution (PBS). The technique selected for the preparation of the MNPs was a double crosslinking (ionic and covalent) process in reverse emulsion which provide the mechanical stability of the polymeric nanocarrier. The chitosan derivative and MNPs were thoroughly characterized by Fourier Transform Infrared Spectroscopy (FT-IR), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM). The Scanning Electron Microscopy photographs revealed that prepared MNPs have different diameters depending on the used stirring rate and polymer concentration. Nanoparticles potential as drug delivery system was analyzed by loading bevacizumab (BEV) a full-length monoclonal antibody. Also, the prepared particles were 1

ACCEPTED MANUSCRIPT found suitable from the cytotoxicity and hemocompatibility point of view enabling their potential use as delivery system for the treatment of posterior segment of the eye conditions. Keywords Poly(ethylene glycol) methacrylate, Chitosan, Double cross-linking, Micro/nanoparticles, Interpenetrating network, Bevacizumab, Ophthalmology. Abbreviations

PT

ABS – Acetate buffer solution AMD – Age-related macular degeneration

RI

BEV – Bevacizumab

SC

CH3COOH – Acetic acid CS – Chitosan CNV – Choroid neovascularization CRVO - Central Retinal Vein Occlusion

NU

CME – Cystoid macular edema

MA

CS-g-PEG - Chitosan grafted poly(ethylene glycol)

CS-g-PEGMA– Chitosan grafted poly(ethylene glycol) methacrylate

D

DS – Degree of substitution

Eq. - Equation

PT E

DSC - Differential scanning calorimetry

FTIR – Fourier Transform Infrared Spectroscopy GA – Glutaraldehyde

CE

GlcN – 2-amino-2-deoxy-β-d-glucopyranosyl GlcNAc – 2-acetamido-2-deoxy-β-d-glucopyranosyl

AC

LEV – Levofloxacin

LPS – Lipopolysaccharide MeO-PEG-g-CS - Methoxy poly(ethylene glycol) acid grafted chitosan mPEG-g-CS - Methoxy poly(ethylene glycol)-grafted-chitosan MNPs – Micro/nanoparticles MPs – Microparticles Mw – Weight average molecular weight NPs – Nanoparticles NMR – Nuclear Magnetic Resonance PBS – Phosphate buffer solution 2

ACCEPTED MANUSCRIPT PDR - Proliferative diabetic retinopathy PEG – Poly(ethylene glycol) PEGMA– Poly(ethylene glycol) methacrylate RBC – Red blood cells rpm - Rotations per minute RT – Room temperature SEM – Scanning Electron Microscope

PT

Span 80 – Nonionic surfactant TGA – Thermo Gravimetric Analysis

RI

Tween 80 – Nonionic Surfactant

SC

TPP - Sodium tripolyphosphate VEGF – Vascular endothelial growth factor

NU

bFGF – Basic fibroblast growth factor 1. Introduction

MA

Disorders affecting posterior eye segment like age-related macular degeneration (AMD), diabetic macular edema, viral retinitis, proliferative vitreoretinopathy, posterior uveitis, retinal

D

vascular occlusions, choroid neovascularization (CNV), and proliferative diabetic retinopathy (PDR) are increasing every year at an alarming rate [1–3].

PT E

Generally, drug delivery to the posterior segment is limited due to the difficulty in delivering effective exact doses of drugs to target tissues within the eye. The administration of pharmaceutical agents may be delivered to the eye through four primary pathways of

CE

administration: topical, systemic, intravitreal and periocular [1]. Current, proven treatment regimens for ocular neovascularization include ocular

AC

photodynamic therapy and laser photocoagulation to either directly treat the CNV as in AMD, or ablate the ischemic retina sparing the macula and non-ischemic areas as in diabetic retinopathy,

vein

occlusions,

retinopathy

of

prematurity

and

anterior

segment

neovascularization. Antiangiogenic therapy relies on a different approach to treat neovascular diseases. The treatment directly targets the angiogenic cascade that it is thought to be initiated by several growth factors such as vascular endothelial growth factor (VEGF), platelet derived growth factor, transforming growth factor, and basic fibroblast growth factor (bFGF). The advantage of antiangiogenic therapy is its potential to preserve the function of retinal tissue while directly targeting the neovascular complexes. The antiangiogenic drugs that are currently known as effective are: monoclonal antibodies that target specific molecular sites; 3

ACCEPTED MANUSCRIPT bevacizumab (Avastin) - the “parent” molecule of ranibizumab, a full-length monoclonal antibody, corticosteroids (intravitreal triamcinolone acetonide, dexamethasone, fluocinolone acetonide, anecortave acetate) [4, 5]. Bevacizumab (BEV) was authorized in 2004 for the chemotherapy treatment of metastatic colorectal cancer [6], in 2006 for cell lung cancer [7], in 2009 for glioblastoma multiform [8]. Intravitreal injections of BEV have been used for the treatment of several eye disorders such

PT

as AMD, central retinal vein occlusion (CRVO), PDR, rubeosis iridis, pseudophakia cystoid macular edema (CME), CNV, secondary to pathologic myopia. Studies in vivo revealed the absence of ocular toxicity for BEV administrated by intravenous injection. Due to its short half-

RI

life and the necessity of frequent intravitreal injection, a method for sustained delivery is

SC

required [9–12].

Studies concerning BEV usefulness in the field of ophthalmology as an off-label drug for the

NU

treatment of wet AMD have been reported. Abrishami et al. study reported that liposome loaded with BEV presented a beneficial effect such as sustained release in the vitreous in animal model during 42 days [13].

MA

Nano systems based on polymeric biomaterials have been extensively investigated because of their numerous advantages, such as protecting sensitive drug molecules from degradation,

D

controlled release properties, and increasing the bioavailability of poorly water-soluble drugs [14].

PT E

Hao et al. have prepared nanoparticles (NPs) based on poly(D,L-lactide-co-glycolide)-loaded BEV, capable to have a prolonged release for 4 weeks [15]. Also, Pan and co-workers have successfully performed two long-lasting formulation based on

CE

poly(ethylene glycol) and poly (lactic-co-glycolic acid). Authors obtained NPs based on poly(lactic-co-glycolic acid)-encapsulated BEV via solid-in-oil-in-water encapsulation method

AC

and poly(ethylene glycol)-BEV conjugate for the treatment of CNV. Both formulations presented a long and sustained release of BEV during 8 weeks [16]. Micro/nanospheres were prepared from poly(D,L-lactide-co-glycolide) and poly(ethylene glycol)-b-poly(D,L-lactic acid) for the treatment of AMD by Fengfu and colobarotors. The study results revealed that BEV loaded micro/nanospheres could be released in a controlled sustained manner over 90 days. The drug release rate could be adjusted by modification of the drug/polymer ratio. Study results indicate that this type of carrier’s system loaded with drug could improve the treatment of wet AMD and cancer [17]. Also, the use of cationic polysaccharides such as chitosan and its derivatives as polymeric matrix for drug delivery generate particular interest due to their unique physicochemical and 4

ACCEPTED MANUSCRIPT biological properties. Chitosan (CS) is a natural biopolymer obtained by alkaline deacetylation of chitin, an abundant biopolymer isolated from the exoskeleton of crustaceans, such as shrimps. This natural amino polysaccharide stands out due to its properties like biocompatibility, biodegradability, mucoadhesivity [14] and characteristics such as antibacterial, anti-tumor, hemostatic, low toxicity and immunogenicity, fungal, analgesic, and film-forming, pH sensitivity. It is suitable to easily prepare targeted drug-delivery nanocarriers

PT

[18–20]. Still, the most important obstacle associated with the use of CS for improvement of drug absorption is its limited solubility at pH higher than its apparent pKa of 6.5, meaning is only

RI

soluble in some dilute acid solutions such as acetic acid and hydrochloric acid. Thus, a various

SC

number of different chemical modification for improving chitosan solubility in water such as sulfonation, quaternarization, carboxymethylation, N- and O-hydroxyalkylation, modification

NU

with thiol groups, graft copolymerization etc., have been reported [21]. Chemical modification of CS via graft copolymerization is a promising method and provides a wide variety of molecular characteristics. Grafting hydrophilic natural or synthetic polymers

MA

onto CS backbone not only enhances the water solubility of the polysaccharide, but also allows the preparation of particles with good stability in dilute conditions [22].

D

Poly(ethylene glycol) (PEG) is a linear polyether diol, a important synthetic macromolecule, frequently used as a non-ionic hydrophilic polymer in bio-science and technology. PEG

PT E

presents a «camouflage behavior» in the case of conjugation with drugs, it protects them from recognition by the immune system and prolongs contact time with tissue and efficacy (bioavailability). PEG is an excellent graft forming polymer because is soluble in water and

CE

organic solvents, and possess thermo responsive properties, low toxicity, good biocompatibility and biodegradability [23].

AC

Nevertheless, particular attention has been dedicated to modified chitosan with PEG (CS-gPEG), the grafting process usually being performed on the amino group with various approaches available.

Generally, one of the CS-g-PEG advantages is the water solubility in a wide range of pH from 1.0 to 11.0, depending on the substitution degree [24]. Nanoparticulate systems-based CS-gPEG deals with the main problems, such as rapid clearance from the blood stream through phagocytosis after intravenous administration and recognition by the macrophages of the mono-nuclear phagocyte system [23]. PEGylation of natural or synthetic polymers or drugs benefits of nontoxicity, reduced clearance by the reticuloendothelial system, a prolonged circulation time in the blood flow, and 5

ACCEPTED MANUSCRIPT an increased stability of the drug against enzymatic degradation and reduce its immunogenicity [24, 23]. Nanocarriers based on CS grafted PEG, have been investigated as potential applications in drug delivery [25]. Due to the presence of unmodified amine groups PEGylated chitosan have proprieties such as self-aggregation (by strong inter- or intra-molecular hydrogen bonds) or complexation (by electrostatic interaction with negatively charged compounds), which makes

PT

them suitable materials for potential applications in drug delivery [24]. Methods used to obtain nanoparticulate systems-based CS-g-PEG are self-aggregation [26], complexation [25], ionic gelation technique [27–30], solvent evaporation method [31].

RI

For example, Ouchi et al. prepared a copolymer methoxy poly(ethylene glycol) acid grafted

SC

chitosan (MeO-PEG-g-CS) which spontaneously form nanosized (<120 nm) aggregates in aqueous solution by strong intermolecular hydrogen bonds between CS moieties. Authors have

NU

investigated the aggregation phenomenon of the copolymer [26]. Bae et al. have synthesized CS-g-PEG/heparin a self-assembly polyelectrolyte complex for inducing apoptotic death of cancer (B16F100) cells in vitro. The micelles (200 nm) obtained

MA

were stable under physiological environment due to the PEG shell layer [32]. Other study performed by Yang et al. shows the ability of methoxy poly(ethylene glycol)-

D

grafted-chitosan (mPEG-g-CS) to self-aggregate and forming nanosized carriers (300 nm) with loading efficiency depending of the degree of substitution (DS) [25].

PT E

Zhang et al. performed a poly(ethylene glycol)-grafted-chitosan which was used for prepare NPs (>300 nm) by the ionic gelation method using sodium triphosphate (TPP) as cross-linker. The NPs were loaded with insulin and presented a loading efficiency of 38%. The study results

CE

showed that the NPs diameter and insulin release from mPEG-g-CS/TPP/insulin NPs could be controlled just by modifying the composition or temperature [28].

AC

Herein, we decided to uncover the particularities of novel micro/nanoparticles (MNPs) – based polymer systems for the transport, targeting and controlled release of drugs used to treat diseases of posterior segment of the eye. Thus, in the present work, we study the synthesis of a new polymer chitosan grafted poly(ethylene glycol) (CS-g-PEGMA) methacrylate and also, the preparation of a micro/nanoparticulate system. The strategy involves the use of CS-gPEGMA with high solubility in water, obtained via Michael addition, in order to prepare potentially non-toxic MNPs. First, the synthesis and thorough characterization of the polymer and the methods used for CS-g-PEGMA characterization (Fourier Transform-Infrared Spectroscopy analysis (FT-IR), Nuclear magnetic resonance (NMR), Thermo Gravimetric Analysis (TGA), Differential scanning calorimetry (DSC)) have been described. Secondly, the 6

ACCEPTED MANUSCRIPT obtained MNPs characteristics such as size, polydispersity, the degree of swelling can be controlled by changing the reaction parameters (water/oil ratio, polymer concentration, dispersion speed, amount of cross-linking agent, the viscosity of the two phases, etc.). The MNPs were obtained through a double crosslinking technique (ionic and covalent) applied in reverse emulsion system, previously elaborated by our research group. The MNPs have been analyzed structurally by FT-IR spectroscopy, morphologically by SEM, gravimetrically for

PT

swelling abilities. Also, hemocompatibility and cytotoxicity were performed to test the applicative potential of the particles. Finally, bevacizumab loading and in vitro release are studied.

Also,

the

antiangiogenic

effect

of

selected

MNPs

was

studied

for

RI

diabetes/inflammatory diseases of the eye and central retinal vein occlusion - experimental

SC

animal model.

NU

2. Experimental 2.1. Materials

MA

The following materials were used for the experiments: low molecular weight Chitosan (CS), (degree of deacetylation 82 %, Aldrich); Poly(ethylene glycol) methacrylate (PEGMA, Mn = 500, Aldrich); Acetic acid (99%, Sigma Aldrich); Glutaraldehyde (GA) (Sigma Aldrich, 25%

D

aqueous solution); Sodium tripolyphosphate (TPP, Sigma Aldrich); Sodium sulfate (Na2SO4,

PT E

Sigma Aldrich); Bevacizumab [(BEV); (25mg/ml, Avastin®, Genentech Inc)] was provided by University of Medicine and Pharmacy “Grigore T Popa” Iasi, Romania; Tween 80 (Sigma Aldrich); Span 80 (Sigma Aldrich); Acetate and phosphate buffered saline (ABS and PBS) lipopolysaccharide, E. coli 055:B5 (LPS, Sigma

CE

were purchased from Sigma-Aldrich;

Aldrich); adult rabbits from Baneasa (Romania); Eritrosein B ( ≥95 %, Sigma Aldrich);

AC

Ketamine (Sigma Aldrich); Xylazine (99 %, Sigma Aldrich); Phosphate buffered saline solution Hank (HBSS, Sigma Aldrich); Collagenase / Dispase ( Sigma Aldrich); Bovine serum albumin (Sigma Aldrich); GST tag (26 kDa, Sigma-Aldrich); Milli-Q ultrapure distilled water (Merck) were of analytical grade and used without further purification. The chemicals used in this study were of analytical grade purity and were used without further purification. The human blood samples used were freshly obtained from one healthy nonsmoker volunteer. Fibroblast cells were extracted from rabbit dermis in the Bioengineering Department of “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania.

7

ACCEPTED MANUSCRIPT 2.2. Methods 2.2.1. Synthesis of chitosan grafted poly(ethylene glycol) methacrylate In order to obtain chitosan grafted poly(ethylene glycol) methacrylate (CS-g-PEGMA) via Michael addition reaction, a modified protocol reported by Ma and collaborators was considered [33]. Briefly, chitosan (1.0 g) and 100 mL acetic acid solution 1% (w/w) were added to a 250-ml

PT

reaction flask equipped with a reflux condenser. The reaction flask was immersed into a water bath previously heated at 400 C, purged with nitrogen and allowed to stir for 60 minutes.

RI

Subsequently, PEGMA was added drop wise at a final molar ratio NH2: PEG = 1:2. Afterwards,

SC

Michel addition reaction was carried out at 600 C, under nitrogen atmosphere and maintained for 24 h to complete the reaction. Finally, the polymer solution was filtered by centrifugation,

NU

rotary evaporated, and precipitated into acetone. The polymer was purified by repeated washing with methanol and acetone. The final product was dried in a vacuum oven for 48 h to

MA

constant weight. A light brown powder was obtained. 2.2.2. Preparation of micro/nanoparticles

Micro/nanoparticles (MNPs) based chitosan grafted poly(ethylene glycol) methacrylate were

D

performed by double cross-linking, ionic followed by covalent in reverse emulsion (w/o)

PT E

according to a method reported by C.A. Peptu and coworkers [34–36]. Therefore, a calculated amount of CS-g-PEGMA (depending of desired concentration) derivative was dissolved in CH3COOH solution (1% w/w). An appropriate quantity of nonionic surfactant Tween 80 (2%

CE

w/w) was added in the polymer solution and well homogenized. Afterwards, the mix was added slowly drop wise in toluene containing an adequate amount of surfactant (Span 80) (200 mL),

AC

under vigorous stirring (different stirring speed). Following the emulsion stabilization time, the amount of ionic cross-linker solution (TPP or Na2SO4, 5%) was added, after which the reaction mix was transferred to a mechanical stirring-equipped reactor, were the process of ionic crosslinking continued at 500 rotations per minute (rpm). The covalent cross-linking process, was carried out by adding in the reactor a calculated amount of glutaraldehyde (GA) extracted in toluene (c = 1.12 mg/mL). After crosslinking reaction ended, the emulsion was centrifuged and removed the toluene phase. MNPs obtained were repeatedly washed with distilled water, acetone and hexane, before being dried out at room temperature. After the purification steps MNPs samples were considered for chraracterization: diameter and size distribution, morphology, swelling behavior, drug loading and release, zeta potential, hemocompatibility 8

ACCEPTED MANUSCRIPT analysis, toxicity and cytotoxicity test, etc. The experimental protocol with the observed parameters for the preparation of MNPs and their average diameter are presented in table 1 and 2, with the mention that the water/oil phase ratio was maintained at 1:4 and the CS/GA molar ratio was kept constant at 1:2. Table 1 Table 2

PT

2.3. Characterization of chitosan derivative and micro/nanoparticles

RI

2.3.1. Techniques of characterization used

SC

Fourier Transform Infrared Spectroscopy spectra of CS, PEGMA, CS-g-PEGMA derivative and micro/nanoparticles were recorded with a DIGILAB SCIMITAR FTS 2000 spectrometer.

NU

The samples were prepared as KBr pellets and scanned over the wave number range of 4000– 450cm−1 at a resolution of 4.0 cm−1. 1

H NMR spectra were obtained by using a Bruker Avance DRX 400. CS, PEGMA (Mn=500)

MA

and CS-g-PEGMA derivative were dissolved in D3CCOOD/D2O or D2O according to their solubility. The substitution degree (DS) was calculated from the peak area at about chemical

D

shift of –CH2b– proton against that of NHAc proton.

PT E

Thermo Gravimetric Analysis (TGA) was carried out with a Mettler Toledo model TGA/SDTA 851 (TGA Q 500-1285 system). Dried samples (3 mg) were loaded in an open aluminum pans, heated under a dynamic N2 atmosphere, with a heating rate of 100 C/min, in a temperature range from the room temperature to 8000 C.

CE

Differential Scanning Calorimetry (DSC) was carried out with a Q20 Series™-DSC. Dried samples (3 mg) were analyzed under a N2 atmosphere on the temperature range 250 C–3000 C.

AC

MNPs morphology was investigated by SEM technique. SEM images were recorded with a HITACHI SU 1510 (Hitachi SU-1510, Hitachi Company, Japan) Scanning Electron Microscope, MNPs were fixed on Aluminum stub and coated with a 7 nm thick gold layer using a Cressington 108 device before observation. The NPs mean diameter and size distribution were analyzed by laser light diffractometry technique (SHIMADZU SALD 7001). Aliquots of MNPs samples were immersed in acetone and sonicated for 15 minutes at room temperature using a sonication bath (Bandelin Sonorex). The suspension of MNPs was introduced in a quartz cuvette equipped with a stirring mechanism and analyzed. All measurements were performed in triplicate.

9

ACCEPTED MANUSCRIPT 2.3.2. Nanoparticles behavior in aqueous media In order to predict and understand the behavior of MNPs during loading and release process, swelling studies were performed by gravimetric method. Briefly, 0.030 g freeze-dried samples of MNPs, were weighted and immersed in vials (eppendorfs) containing 1 ml acetate buffer solution (ABS) (pH=3,3) or phosphate buffer solution (PBS) (pH=7,4). The vials were maintained at room temperature under mechanical stirring. Swelling process was monitored

PT

and after preset times, MNPs suspensions were ultra-centrifuged (15.000 rpm) and weighted after supernatant removal. Swelling process was monitored by recording the liquid uptake at

Q% =

𝑤𝑠 −𝑤0

× 100

(1)

NU

where:

𝑤0

SC

swelling ratio was determined with equation (Eq.) (1):

RI

pre-determined time intervals until equilibrium swelling was attained. The percentage of

ws - the weight of swollen probe;

MA

w0 - the weight of dry probe.

2.3.3. Evaluation of bevacizumab loading kinetic

D

Drug loading process was carried out through diffusional mechanism. In this regard, BEV

PT E

was used as model drug. Briefly, 0.030g CS-g-PEGMA NPs dry powder were suspended in 1 ml of BEV aqueous solution (25 mg/mL) and dispersed by sonication for 15 minutes. The suspensions were maintained at room temperature, under gentle mechanical stirring for 72

CE

hours. After preset times intervals samples were centrifuged (at 15.000 rpm, 5 minutes) and the quantity of BEV retained was calculated by determining the drug content from supernatant

AC

based on a calibration curve previously obtained, with the equations from Eq. (2): 𝑦 = 0.1373 ∙ 𝑥; 𝑅 2 = 0.9846

(2)

All tests were accomplished with a spectrophotometer UV–VIS NanoDrop ND -1000, which allows the analysis of very small sample volumes, in microliter range. BEV loaded NPs were freeze-dried. 2.3.4. Bevacizumab in vitro release kinetic Drug release kinetic was performed in PBS (pH = 7.4 similar to aqueous humor of the eye) in order to ensure a lower release rate. The released drug was determined by monitoring the

10

ACCEPTED MANUSCRIPT wavelength at 272 nm for BEV. The release kinetics investigation, was carry out by immersing the BEV loaded NPs in 1.0 mL PBS at 370 C, under continuous gentle stirring (100 rpm), after which the drug concentration in the supernatant was spectrophotometrically determined. All measurements were performed in triplicate and averaged. 2.3.5. Nanoparticles hemocompatibility Hemolysis tests were performed using a method proposed by Vuddanda et al. [37]. First, 5

PT

mL of blood were centrifuged for 5 minutes at 2000 rpm. Supernatant plasma surface layer was removed and the red blood cells (RBC) were separated and washed with normal saline solution.

RI

The purified RBCs were re-suspended in saline solution to obtain 25 ml of RBC suspension.

SC

2 mL of NPs suspension in saline solution at different concentrations were added to 2 mL of RBC suspension (final concentrations were 100 μg NPs/ml, 200 μg NPs/ml and 400μg

NU

NPs/ml). Positive (100 % lysis) and negative control samples (0% lysis) were prepared by adding equal volumes of 2 % Triton X-100 solution and saline, respectively, over the RBC suspension. The samples were incubated at 370 C for 2, 4 and 6 hours. The samples were gently

MA

shaken every 30 minutes for re-suspension. After the incubation time, the samples were centrifuged at 2000 rpm for 5 minutes, and each 1.5 mL of the supernatant was incubated for

D

30 minutes at room temperature to allow hemoglobin oxidation. Oxyhemoglobin absorbance in supernatant was measured with a spectrophotometer (PG Instruments T60 UV-Vis

PT E

Spectrophotometer) at 540 nm. The hemolysis percentages of RBC were calculated by the following Eq. (3). All experiments were performed in triplicate. 𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒−𝐴𝑏𝑠 𝑛𝑒𝑔𝑎𝑡𝑖𝑣 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

(3)

CE

% 𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 = 𝐴𝑏𝑠 𝑝𝑜𝑧𝑖𝑡𝑖𝑣 𝑐𝑜𝑛𝑡𝑟𝑜𝑙−𝐴𝑏𝑠 𝑛𝑒𝑔𝑎𝑡𝑖𝑣 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

2.3.6. Study of antiangiogenic effect of selected NPs on diabetes/inflammatory diseases

AC

of the eye and central retinal vein occlusion - experimental animal model Inflammation of the rabbit eye (5 animals) was induced by injection of lipopolysaccharide intravenously, 10 g/kg/100 l, at 72 hours for 18 days, according to the literature [38]. Rabbit diabetes (5 animals) was induced by streptozotocin injection, 180 mg/kg i.p. in single administration. Demonstration of onset of diabetes was made by sequential measurements of blood glucose after induction, one day, 3, 7, 14 and 21 days [39]. Central retinal vein occlusion (CRVO) in the rabbit (5 animals) was induced by intraperitoneally injection, 20 mg/kg, of Eritrosein B solution (2 %), according to the literature but slightly adapted [40]. 11

ACCEPTED MANUSCRIPT The CRVO was induced within 5 minutes (pre-experimental interval) by focused use of the laser wavelength of 532 nm, laser (Nikon Eclipse TE-300) present in the confocal laser microscopy setup (Microradiance, BioRad). Thus, the laser beam was intraocularly focused (in rabbits) by detaching the conduction tube from the microscope and setting in front of the open eye. One eye was used for the experiment, and the contralateral eye served as control. With such laser (power of 5 mW) 10-second

PT

flashgun train at every 25 seconds was applied to the rabbit’s eye. This was the highest success rate (approximately 95%) obtained. For the experiment, adult rabbits were grown under

conditions and anesthetized and sacrificed (without pain).

RI

standard laboratory conditions, nourished with standard feed for rabbits, treated under human

SC

In the experiments, rabbit retinal endothelial cells were obtained as described. Were used 5 rabbit retinas, immediately after sacrifice, obtained by dissection under the microscope. These

NU

were homogenized in HBSS, on ice, without Ca2 + and Mg2 +. After centrifugation, the sediment was digested for 1 hour at 370 C in the presence of a mixture containing 2 mg/ml collagenase/dispase, DNA type 1 from bovine pancreas (20 µg/ml) and Tosyl lysine

MA

chloromethyl ketone (50 µg/ml) in HBSS and without Ca2+ and Mg2+, but with penicillin, streptomycin and 10 mM HEPES, at pH= 7.4. After digestion, the suspension was filtered

D

through a 70 μm sterile nylon sieve and microvascular structures retained on the filter were washed twice with HBSS and without Ca2+ and Mg2+. In order to reflect the number of

PT E

endothelial cells, the supernatants obtained in separation processes above were aspirated several times through a 1 ml pipette tip for mechanical dispersion and then incubated at 40 C in PBS containing 1 % bovine serum albumin and 20 mg/ml fluorescein bound lectin [41].

CE

After 24 hours, the cells were washed by centrifugation and the free fluorescein removed, and the endothelial cells were quantified by confocal laser microscopy, the HQ515/530 filter was

AC

used as a 488 nm excitation filter. The green fluorescein fluorescence lectin coupled, was quantified to highlight the percentage of the endothelial cells number under conditions of inflammation, diabetes and CRVO in experimental rabbits, already mentioned. Basically, was quantified the number of pixels associated with endothelial cells. The experiments were performed in the presence of simple nanoparticles or loaded with BEV and monoclonal antibody (mouse) anti-VEGF-A human (equivalent bevacizumab) on cells derived from inflammatory, diabetic models and CRVO. The immunogenic sequence (VEGF) is a partially recombinant protein with the GST tag. All types of NPs, whether or not loaded with a biologically active substance, were administered in

12

ACCEPTED MANUSCRIPT suspension (1%), by intraocular injection, for 72 hours until the animals were sacrificed, after the animal model of inflammation, diabetes or CRVO was achieved. 3. Results and discussion 3.1. Characterization of CS-g-PEGMA Chitosan characteristics and properties such as non-toxicity towards living organisms,

PT

biodegradability, hemocompatibility, biocompatibility and the amino and hydroxyl groups make him suitable for chemical modification. The higher reactivity of the amino group

RI

compared to hydroxyl groups, is one of the reasons for which amino groups are the most used in chemical modifications. Published works on CS modified with PEG, report that PEG was

SC

grafted to the amino group of the glucosamine unit. CS was chemically modified through various methods, but reactions such as reductive amination, condensation reaction have

NU

disadvantages including organic medium, complex or toxic catalyst, protection and deprotection of groups, which limits the polysaccharide derivatives utilization in biomedical

MA

filed. Through Michael addition were synthesized few chitin and chitosan derivatives. Michael addition reaction have advantages such as mild reaction conditions, high functional group tolerance, a large host of polymerizable monomers and functional precursors, high conversions,

D

lack of side reactions, favorable reaction rates and versatility in terms of: monomer selection,

PT E

solvent environment, reaction temperature. In present work, CS-g-PEGMA derivative was synthesized through Michael addition of poly(ethylene glycol) methacrylate to the amino

Scheme 1

CE

groups of chitosan. Reaction of chitosan grafted PEGMA (Scheme 1) was carried out at 400 C.

A first structural characterization of the grafted chitosan derivative is given by the Fourier

AC

Transform-Infrared Spectroscopy analysis. FT-IR spectra of CS, PEGMA, CS-g-PEGMA are presented in Fig. 1. The infrared spectrum of the CS showed a strong peak at 3365 cm−1 that could be attribute to the axial stretching vibration of –OH superimposed to the –NH2 stretching band and inter- and extra- molecular hydrogen bonding of CS molecules which decreased in the acylated CS derivatives. The absorption peaks at 1646, 1423 and 1379 cm−1 are due to the NHAc units, the amide I, –NH2 bending and the amide III [42]. The vibration at 1076 cm−1 is characteristic to -C-O-C- stretching and its saccharine structure [43]. The prominent peak at 1726 cm−1 was assigned to double bond of the PEGMA. Two peaks demonstrate that PEGMA was grafted to CS backbone. The first new peak at 1732 cm−1

13

ACCEPTED MANUSCRIPT appeared and is assigned to C=O of -OCOR group. This peak highlights the presence of PEGMA in the structure of the functionalized CS. The second new peak is at 1649 cm-1 and is typical for the composition of the polysaccharide. A slight displacement of the absorption bands for the grafted product comparative with the base-products was observed, the cause being the surrounding groups which influences the wavelength. The signal for the formed amino group cannot be define because the peak signal it is around 3300–3400 cm-1. Both CS and PEGMA have hydroxyl groups and they give a signal around the same value 3300–3400

PT

cm-1 overlapping the secondary amine group (-NH-). The peaks at 2941 and 2879 cm-1 are characteristic for -C-H stretching, but they are not significant because CS and PEGMA give

RI

approximately the same signal. The intensity of absorption peaks at 1646 and 1726 cm-1

SC

decreased due to their participation in reaction. It was confirmed that PEGMA group substituted the amino group. A characteristic band at 3400 cm-1 is attributed to -NH2 and - OH

NU

groups stretching vibration and the band for amide I at 1646 cm-1 is seen in the FT-IR spectrum of modified chitosan.

MA

Fig. 1.

The structural characterization of CS-g-PEGMA was performed also by NMR. The 1H-NMR

modified polymer. The

1

D

recorded spectra offer useful information regarding the chemical composition of obtained H-NMR spectrum of CS, PEGMA and CS-g-PEGMA in

PT E

D3CCOOD/D2O or D2O at 800 C is shown in Fig. 2 a–c. A small peaks at 1.795 ppm existed because of the presence of -CH3 of N-alkylated GlcN residue. A singlet at 3.139 ppm assigned to H2 of 2-amino-2-deoxy-β-d-glucopyranosyl (GlcN) and N-alkylated GlcN. The multiplets

CE

from 3.5 to 3.8 ppm attributed to H3, H4, H5, H6 of GlcN and N-alkylated GlcN. A small peak at 4.52 ppm attributed to H1 of GlcN and N-alkylated GlcN. While a small signal of formed

AC

methylene protons adjacent to the 2-amino group of the GlcN residue was observed at 1.994 ppm (-CH2b-COO-). The typical peak at 4.528 ppm is appeared because of -CH2d of GlcN residue. The DS values based on -CH2b-COO- was determined by the relative intensities of these signals and was calculated from the peak area of CH2b- proton against NHAc proton. The DS calculation was calculated according to the Eq. (4): 𝐷𝑆 =

3∙𝐼3.1 𝑝𝑝𝑚 ∙(1−𝐷𝐷) 2∙𝐼2.0 𝑝𝑝𝑚

(4)

The calculated DS for CS-g-PEGMA was 11.2%. Fig. 2. 14

ACCEPTED MANUSCRIPT Fig. 3 a) and b) shows the TGA analysis of CS and CS-g-PEGMA. CS thermogram highlights the existence of three important stages. The first stage is an endothermic peak appeared at 61.560 C which is specific to water evaporation and the mass loss of 7.22 % of the sample. The second stage is highlighted by the appearance of other endothermic peak at 258.430 C is attributed to the decomposition of CS. The exothermic peak appeared at 345.240 C marks the third stage of the CS thermogram and corresponds to thermal decomposition of the polymer.

PT

The thermogram of the grafted product (Fig. 3b) shows that after being subjected to a thermal degradation the sample presents weight loss due to the degradation of CS and PEGMA. As we have noted from the CS thermogram, the CS-g-PEGMA thermogram has also three stages. The

RI

first and second stage highlighted by CS-g-PEGMA thermogram corresponds to loss of water,

SC

moisture content of the polysaccharide and to the decomposition of CS-g-PEGMA. The broad exothermic peak situated at 648.330 C corresponds to CS-g-PEGMA thermal decomposition.

NU

These results suggested that the structure of chitosan chains has been changed due to the introduction of PEGMA. The degradation stages according to TGA for pure chitosan, poly(ethylene glycol) methacrylate and CS-g-PEGMA, under an atmosphere of nitrogen, are

MA

presented in table 3. Table 3.

D

Fig. 3.

PT E

3.2. Characterization of micro/nanoparticles Micro/nanoparticles based CS-g-PEGMA were performed by double cross-linking, ionic followed by covalent, in reverse emulsion (w/o). After the purification steps freeze dried MNPs

CE

samples were considered for different types of chraracterization. First, FT-IR analysis for MNPs revealed the formation of the polymer network through double

AC

crosslinking. FT-IR spectra were recorded for all the MNPs obtained, but they showed a similar profile. As representative example, only the FT-IR spectra for samples MA-8 and MA-H are displayed (Fig. 4), the difference being the nature of the ionic crosslinker. Table 4 shows the characteristics absorption band of chitosan derivative and samples MA-8, MA-H. From the spectra samples outlined in Fig. 4, we can see for sample MA-8 the appearence of a new signal at 891 cm-1 which correspond to the new bond formed due to the ionic crosslinking process, between tripolyphosphate anions and CS-g-PEGMA ammonium cations.

15

ACCEPTED MANUSCRIPT Also, in the case of the sample MA-H, also we observed the appearence of a signal at 636 cm1

which correspond to the new bond formed due to the ionic crosslinking process, between the

sulfate anions from Na2SO4 and the ammonium cations of CS-g-PEGMA. The absorption bands of imine linkages -C=N- resulting from the covalent crosslinking process are highlighted at 1541 cm-1 for sample MA-8 and 1548 cm-1 for sample MA-H. The covalent crosslinking process took place between the amine groups of the chitosan derivative

PT

and the carbonyl groups of glutaraldehyde [44–49]. Fig. 4.

RI

Table 4.

SC

MNPs were analyzed also by DSC. According to the curves presented in the DSC thermograms presented in Fig. 5, it can be seen that the melting process shows an endothermic

NU

peak of CS which is high at 94.010 C, for chitosan derivative at 102.560 C and the NPs at 106.20 C. By comparing the results obtained, can be affirmed that the NPs thermogram is similar to CS and CS-g-PEGMA thermogram, suggesting a good thermal stability of NPs. Also, the

MA

appearance of the exothermic peak at 253.750 C indicates the formation of new chemical bonds in the NPs structure, that can be attributed to ionic and covalent crosslinking processes. Also,

D

results presented in Fig. 5 indicates that NPs based CS-g-PEGMA obtained are stable.

PT E

Fig. 5.

Furthermore, CS-g-PEGMA NPs morphology was investigated by SEM technique. Scanning electron microscopy images for different MNPs samples indicates the fact that after

CE

optimization steps, the NPs obtained presented a spherical shape, are individualized and the best sample crosslinked with TPP (MA-7, 8) have size ranged from 200 nm to 900 nm in

AC

diameter, also the polydispersity is reduced. In the case of samples (MA-G, H) crosslinked with Na2SO4 the size is ranging from 1000 to 1500 nm due to a low crosslinking density. SEM photographs for the most representative samples are presented in Fig. 6, 7. For samples MA-8–11 and MA-H–K the polymer/crosslinker ratio and the stirring rate were kept constant, only the polymer concentration was modified. It was observed that the particle diameter is dependent of the polymer concentration. The highest diameter and polydispersity, respectively a lower agglomeration tendency was recorded in the case of MA-11, respectively MA-K sample, polymer concentration being 0.75%, this behavior was reported also, in other studies [34, 35]. An explanation being the viscosity of the polymer solution which increases when the concentration is higher, leading to big droplets in the emulsion phase wich need to be 16

ACCEPTED MANUSCRIPT crosslinked. After subtracting the polymer concentration to 0.3%, was observed the formation of irregular particle (MA-9 and I ), a possible cause a weaker cross-linking process. In the case of MA-10 and J particles, the polymer/ionic crosslinking ratio was modified, and led to a small increasing of particle diameter, due to the increment of the cross-linking density of the polymer matrix. Another important aspect highlighted by SEM photographs is that the diameter and

PT

polydispersity of the particles is influenced by the nature of the crosslinking agent. Thus, the change of the ionic crosslinker in the case of MA-A–K samples, was used sodium sulphate fact

RI

which leads to an increased particle diameter, according to our expectations. Fig. 6.

SC

Fig. 7.

Also, laser light diffractometry measurements brought important information about MNPs

NU

size and polydispersity, which was confirmed also by SEM results. Analysis results indicated that polymer concentration and stirring intensity influences the NPs diameter. As expected, for

MA

both types of MNPs the diameter decreased with the increase of the stirring speed above 9000 rpm, the smaller mean particle size may be attributed to a greater cross-linking density. Also, the results confirmed that the diameter of the both types of particles is reduced as the initial

D

polymer concentration in the synthesis decreased. Regarding the dimensional polydispersity

PT E

curves (Fig. 8–11) they have unimodal aspect and MNPs size are in concordance with the SEM images previously presented. All measurements were performed in triplicate.

Fig. 10. Fig. 11.

AC

Fig. 9.

CE

Fig. 8.

In order to evaluate a very important factor the NPs ability to encapsulate/entrap drugs, further characterization of NPs samples was performed by studying swelling behavior in different physiological environments. Swelling behaviors of NPs samples were performed in solutions with different pH values (pH 3.3 and pH 7.4; these pH values were chosen as the suspensions are intended to be used in biological fluids) by recording the liquid uptake at pre-determined time intervals until equilibrium swelling was reached. As expected the results obtained revealed a correlation between the maximum value of swelling degree and MNPs preparation parameters such as stirring speed, polymer concentration (Fig. 12, 13). All MNPs samples were 17

ACCEPTED MANUSCRIPT displaying a higher swelling degree in acetate buffer (pH 3.3) between 700–1100%, compared with those in phosphate buffer (pH 7.4) between 450–650%. We can therefore state that this effect of a higher swelling degree of the MNPs in acidic environment involves the protonation of amino/imine groups which leads to strong electrostatic repulsion between the polymer chains, thus to an increased capacity of the network to include a higher amount of water. Increasing the stirring speed from 5000 to 15000 rotations per minutes (rpm) lead to the

PT

formation of small particle size, suitable for ocular applications, with well-individualized shape, nanometric size and narrow polydispersity, confirmed by SEM and SALD analysis. Fig. 12 present the influence of stirring speed on the swelling process. Thus, appears that the values

RI

of maximum swelling degree are increased by increasing the intensity of stirring.

SC

The results obtained for the swelling degree revealed that by increasing the stirring speed up to 15000 rpm, while maintaining constant the polymer concentration the best value (1100%)

NU

obtained was for sample MA-8.

As we can observe from Fig. 13 by increasing the polymer concentration solution, while maintaining constant the stirring speed leads to a series of changes. Although a lower polymer

MA

concentration (0.35%) was used for MA-9 and MA-I samples, the swelling degree was highest. Subtracting the polymer concentration is correlated with a decreased number of amine groups

D

participating to cross-linking processes, thus a decreased crosslinking density. As a result, a high value for the swelling degree is a logical consequence. Also, was observed that the formed

PT E

particles at 0.75 % polymer concentration, with a higher diameter retained a lower water quantity therefore, a lower swelling degree. The obtained values can be related with a higher agglomeration tendency of the particles and a higher degree of crosslinking.

CE

Also, lower values of swelling degree (MA-10, MA-J) were recorded when the amount of TPP or sodium sulfate was increased, we can state that the result is a natural one due to the

AC

increase in crosslinking density. For all samples, the obtained values for the swelling degree in both aqueous medium is not affected by the ionic crosslinker nature, the experimental values obtained for both acidic and basic media are quite close, the differences being very small. Fig. 12. Fig. 13. NPs stability in aqueous suspension was evaluated by Zeta potential measurements. Table 5 summarizes the arithmetic average of zeta potentials determined by Smoluchowski equation for optimized CS-g-PEGMA MNPs prepared. A large negative or positive zeta potential for 18

ACCEPTED MANUSCRIPT suspended particles indicates that the system is stable against flocculation or coagulation. In general, literature studies report that zeta potentials values for MNPs prepared using grafted copolymers have a tendency to be higher compared to those synthesized only from chitosan, this could indicate a low stability of aqueous suspension of synthesized MNPs. Table 5. The hemolytic potential of MNPs has been studied. Hemolysis is the destruction of red blood

PT

cells to release hemoglobin and other components in the surrounding fluid. Dangerous pathological conditions can be induced by an increase in red blood cell degradation. Therefore,

RI

all products intended for intravenous administration biomedical must be assessed to determine

SC

their hemolytic potential [50]. The MNPs obtained can be used as drug delivery systems and can be administered intravenously and therefore preliminary tests were conducted to determine their interaction with human blood components. The hemolytic potential of NPs based CS-g-

NU

PEGMA was evaluated for concentrations between 100 and 400 mg/mL, using a spectrophotometric method [51, 52]. NPs based CS-g-PEGMA obtained ranging in size

MA

between 200 and 900 nm and positively charged, were demonstrated to be hemocompatible for concentrations less than 50 mg/ml. The results of hemolysis tests are expressed as average ±

D

SD (n = 3) in Fig. 14.

PT E

Fig. 14.

If hemolysis percentage is less than 5%, thus the analysed samples can be considered hemocompatible [53]. For all concentrations tested was obtained a percentage of less than 5%

CE

hemolysis (Fig.14), which indicates that the MNPs are hemocompatible and can be administered intravenously.

AC

The quantitative analysis of the cellular toxicity of two MNPs formulations were also evaluated using the arithmetic method Spearman – Karber, Eq. (5). The average values of LD50 for samples MA-5 is 4922 mg/kg and for MA-8 is 6744 mg/kg. The results shown in table 5, demonstrate that both formulation are nontoxic and can be used in biomedical applications. 𝐿𝐷50 = 𝐿𝐷100 ∙ [∑

a∙b n

]

(5)

where: LD50 - lethal dose a - the difference between two consecutive doses; b - the average number of dead animals from two successive groups; n - the number dead animals from a group; 19

ACCEPTED MANUSCRIPT LD100 - the amount of substance that kills 100% of animals tested. Table 6. The biomaterial character of the synthesized particles was evaluated via cytotoxicity test on osteoblastic cell cultures. Few MNPs samples were selected and the results are shown in Fig. 15. Colorimetric cytotoxicity assay, MTT was performed on the osteoblastic cell cultures type at intervals of 24, 48, or 72 hours. Examination of cell morphology was performed using the

PT

Bouin blending method and hematoxylin and eosin staining of control and experimental cell cultures. Colored formulations were visualized on a microscope (the resolution of the lenses

RI

being 10x and 40x) as shown in Fig. 15. The assay confirmed that the MNPs have a reduced

SC

dose of toxicity a fact also demonstrated by LD50 test. After 5 days, cells have proliferated normally in the presence of MNPs forming a dense cell monolayer (Fig. 15). The cell viability

NU

was 100 % in the case of MA-5 sample, respectively above 80 % in the case of MA-6 and MA8, the increased cell viability indicates that cells have proliferated favorable in the presence of

MA

MNPs compared to control sample, and that MNPs based CS-g-PEGMA are not cytotoxic. Fig. 15.

D

Considering the purpose of using MNPs as a possible drug delivery system for the treatment of posterior segment of the eye conditions, drug loading/release potential has been studied. The

PT E

drug type selection was made considering the MNPs hydrogel character and encapsulation/release diffusion principle. Furthermore, the diffusional process for the MNPs loaded drug in aqueous solution follows the swelling behavior. Taking into account the

CE

morphological analysis, sample MA-8 was selected for the inclusion of BEV. The results obtained prove that the NPs retained the maximum amount of drug (0.327 mg BEV /mg NPs)

AC

after 72 h, with a encapsulation efficiency of 39 %. Fig. 16 a) presents release kinetics of BEV (MA-8 sample) in phosphate buffer, pH = 7.4. The evolution of the BEV release is almost linear, excepting the reduced burst effect at 30 minutes. One can also observe that 51 % of BEV has been released in 168 hours, fact which is suggesting a slow release rate. Fig. 16. Regarding the releasing process of the biological active principles, literature studies specify that it is dependent on factors such as particles preparation method, dimension and, last but not

20

ACCEPTED MANUSCRIPT the least, the physicochemical properties of the used polymers. Also, the analysis of the release kinetics was accomplished based on the Korsmeyer-Peppas mathematical model, Eq. (6). Mt M∞

= k ∙ t n (6)

where: t is the time Mt/M∞ - drug fraction released at time t

PT

k is a kinetic parameter that characterizes the interactions between drug and polymer, n is the parameter which provides information on the type of transport and release

RI

mechanism of the drug through the polymer matrix and was determined from experimental data.

SC

The equation obtained from processing the release kinetics data in the 0 –300 minutes interval is:

NU

y = 0.4968·x – 3.8964 from which were determined the parameters k = 0.053, n = 0.4968 and R2 = 0.9853.

MA

It is known that the value of n for a normal Fickian diffusion is 0.5 < n < 1. The value of the exponent n, quite close to 0.5 indicate that we have a Fickian diffusion (Fig. 17), the drug

D

transport process through the polymer matrix being dominated by diffusion [54–56].

PT E

Fig. 17.

The study of antiangiogenic effect of selected NPs on diabetes/inflammatory diseases of the eye and central retinal vein occlusion (CRVO) - experimental animal model was also

CE

performed. The data obtained from the models of inflammatory diseases, diabetes and CRVO on animals (rabbits) is the basis for the extension of research in terms of combined in vivo

Table 7. Fig. 18.

AC

models by applying the antibodies to the target tissue reactivity as close as possible.

Fig. 19. Fig. 20. As can be seen from table 7 and Fig. 18–20 we can state that the 1% suspension of NPs loaded BEV and monoclonal antibody (mouse)anti-VEGF-A human (equivalent BEV) are very efficient as anti-angiogenic treatment in the case of the model of diabetes in rabbits. Due to the fact that there are no significant differences between the obtained results, both NPs suspensions 21

ACCEPTED MANUSCRIPT could also be used as anti-angiogenic treatments with moderate efficacy in the case of inflammation with lipopolysaccharide and central retinal vein occlusion models in rabbits. 4. Conclusions In this study new polymer chitosan grafted poly(ethylene glycol) methacrylate (CS-gPEGMA) was synthesized by Michael addition reaction, as a new micro/nanocarrier material for drug delivery. The chemical structure of CS-g-PEGMA was confirmed by NMR and FT-

PT

IR analysis. Newly synthesized CS-g-PEGMA, presented an improved solubility in aqueous solution. Micro/nanoparticles based CS-g-PEGMA were prepared through a double

RI

crosslinking technique (ionic and covalent) applied in reverse emulsion system. The SEM

SC

analysis of the MNPs indicated a relative reduced polydispersity, confirmed by laser diffractometry, but an uniform round shape. The NPs dimension ranged from 200 to 900 nm

NU

and they manifested a pH sensitive behavior in aqueous environments. Moreover, in vitro release study indicated that BEV was released from the nanoparticles in a controlled-release manner for several days and is related to the swelling behavior in aqueous environment. NPs

MA

toxicity was similar to the individual polymers, practically nontoxic degree of toxicity. The hemocompatibility tests showed that the NPs are hemocompatibile and can be administered by

D

local injection. Therefore, we believe that MNPs based CS-g-PEGMA could be a potential carrier for controlled release of BEV as an ophthalmic drug delivery system. The major benefits

PT E

of bevacizumab loading in micro/nanoparticles would be: a much lower dose to administer (thus reducing the adverse effects of drugs), their prolonged release (less frequent administration), and the effectiveness of local delivery (which may extend up to at 14-30 days),

CE

their targeted administration by local injection near the retina. Author Information

AC

Corresponding Authors

*E-mail: [email protected] *E-mail: [email protected] Mailing address: Department of Natural and Synthetic Polymers, Faculty of Chemical Engineering and Environmental Protection, “Gheorghe Asachi” Technical University of Iaşi, Romania Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

22

ACCEPTED MANUSCRIPT Notes The authors declare no competing financial interest. References [1] D.H. Geroski, Henry F. Edelhauser, Drug Delivery for Posterior Segment Eye Disease, Invest Ophthalmol Vis Sci. 41 (5) (200) 961–964.

PT

[2] C.A. Peptu, M. Popa, C. Savin, R.F. Popa, L. Ochiuz, Modern Drug Delivery Systems for Targeting the Posterior Segment of the Eye, Curr Pharm Des. 21 (42) (2015) 6055–

RI

69.

[3] E. Sánchez-López, M. Espina, S. Doktorovova, E.B. Souto, M.L. García, Lipid

SC

nanoparticles (SLN, NLC): Overcoming the anatomical and physiological barriers of the eye – Part I – Barriers and determining factors in ocular delivery, Eur. J. Pharm.

NU

Biopharm. 110 (2017) 70–75.

[4] Y. Tamaki, Novel approach for management of age-related macular degeneration

MA

antiangiogenic therapy and retinal regenerative therapy, Nippon Ganka Gakkai Zasshi. 111 (3) (2007) 232–268, discussion 269.

[5] S.A. Teich, T.W. Cheung, A.H. Friedman. Systemic antiviral drugs used in

D

ophthalmology, Surv Ophthalmol. 37 (1) (1992) 19–53.

PT E

[6] N. Ferrara, K.J. Hillan, H.P. Gerber, W. Novotny, Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer, Nat Rev Drug Discov. 3 (5)

CE

(2004) 391–400.

[7] M.H. Cohen, J. Gootenberg, P. Keegan, R. Pazdur, FDA drug approval summary: bevacizumab (Avastin®) plus carboplatin and paclitaxel as first-line treatment of

AC

advanced/metastatic recurrent nonsquamous non-small cell lung cancer, Oncologist. 12 (6) (2007) 713–718. [8] M.H. Cohen, Y.L. Shen, P. Keegan, R. Pazdur, FDA drug approval summary: bevacizumab (Avastin ®) as treatment of recurrent glioblastoma multiforme, Oncologist. 14 (11) (2009) 1131–1138. [9] R.P. Manzano, G.A. Peyman, P. Khan, M. Kivilcim, Testing intravitreal toxicity of bevacizumab (Avastin), Retina 26 (3) (2006) 257–261.

23

ACCEPTED MANUSCRIPT [10] J. Shahar, R.L. Avery, G. Heilweil, A. Barak, E. Zemel, G.P. Lewis et al. Electrophysiologic and retinal penetration studies following intravitreal injection of bevacizumab (Avastin), Retina 26 (3) (2006) 262–269. [11] S.J. Bakri, J.D. Cameron, C.A. McCannel, J.S. Pulido, R.J. Marler, Absence of histologic retinal toxicity of intravitreal bevacizumab in a rabbit model, Am. J. Ophthalmol. 142 (1) (2006) 162–164.

PT

[12] M. Lüke, M. Warga, F. Ziemssen, F. Gelisken, S. Grisanti, T. Schneider, C, Lüke, M. Partsch, K.U. Bartz-Schmidt, P. Szurman, Effects of bevacizumab on retinal function in

RI

isolated vertebrate retina, Br. J. Ophthalmol. 90 (2006) 1178–1182.

[13] M. Abrishami, S. Zarei-Ghanavati, D. Soroush, M. Rouhbakhsh, M.R. Jaafari, B. Preparation,

characterization,

and

SC

Malaekeh-Nikouei,

in

vivo

evaluation

of

nanoliposomes-encapsulated bevacizumab (avastin) for intravitreal administration,

NU

Retina 29 (5) (2009) 699–703.

[14] M. Prabaharan, Review Chitosan-based nanoparticles for tumor-targeted drug delivery,

MA

Int. J. Biol. Macromol. 72 (2015) 1313–1322.

[15] Y. Hao, A. Patel , W. Liu, R. Krishna, N.R. Sabates, A.K. Mitra, Preparation and characterization of bevacizumab (avastin) nanoparticles for the treatment of age related

D

macular degeneration, American Association of Pharmaceutical Scientists (AAPS)

PT E

Annual Meeting and Exposition (2009). [16] C.K. Pan, C. Durairaj, U.B. Kompella, O. Agwu, S.C. Oliver, H. Quiroz-Mercado, N. Mandava, J.L.Olson, Comparison of long acting bevacizumab formulations in the

CE

treatment of choroidal neovascularization in a rat model, J. Ocul. Pharmacol. Ther. 27 (3) (2011) 219–224.

AC

[17] F. Li, B. Hurley, Y. Liu, B. Leonard, and M. Griffith, Controlled Release of Bevacizumab Through Nanospheres for Extended Treatment of Age-Related Macular Degeneration, Open Ophthalmol. J. 6 (2012) 54–58. [18] I.P. Kaur, S. Kakkar, Nanotherapy for posterior eye diseases, J. Control. Release 193 (2014) 100–112. [19] J. Chan, G.M.M. El Maghrabya, J.P. Craig, R.G. Alany, Phase transition water-in-oil microemulsions as ocular drug delivery systems: In vitro and in vivo evaluation, Int. J. Pharm. 328 (1) (2007) 65–71.

24

ACCEPTED MANUSCRIPT [20] I. Capek, Preparation of netal nanoparticles in water- in- oil (w/o) microemulsions, Adv. Colloid. Interface Sci. 110 (1-2) (2004) 49–74. [21] J. Desbrieres, C.A. Peptu, C. Savin, M. Popa, Chemically modified polysaccharides with applications in nanomedicine, in Biomass as renewable raw materials for bioproducts, V. a. I.Volf Ed., Elsevier (2018) 372–399. [22] D.-J. Fu, Y. Jin, M.-Q. Xie, Y.-J. Ye, D.-D. Qin, K.-Y. Lou,Y.-Z. Chen, F. Gao,

PT

Preparation and characterization of mPEG grafted chitosan micelles as 5-fluorouracil carriers for effective anti-tumor activity, Chin. Chem. Lett. 25 (11) (2014) 1435–1440.

RI

[23] S.A. Papadimitriou, D. S. Achilias, D. N. Bikiaris, Chitosan-g-PEG nanoparticles ionically crosslinked with poly(glutamic acid) and tripolyphosphate as protein delivery

SC

systems, Int. J. Pharm. 430 (1-2) (2012) 318–327.

[24] L. Casettari, D. Vllasaliu, E. Castagnino, S. Stolnik, S. Howdle, L. Illum, Review

NU

PEGylated chitosan derivatives: Synthesis, characterizations and pharmaceutical applications, Prog. Polym. Sci. 37 (5) (2012) 659–685.

MA

[25] X. Yang, Q. Zhang, Y. Wang, H. Chen, H. Zhang, F. Gao, L. Liu, Self aggregated nanoparticles from methoxy poly(ethylene glycol)-modified chitosan: synthesis; characterization; aggregation and methotrexate release in vitro, Colloids Surf B. 61 (2)

D

(2008) 125–131.

PT E

[26] T. Ouchi, H. Nishizawa, Y. Ohya, Aggregation phenomenon of PEG grafted chitosan in aqueous solution, Polymer 39 (21) (1998) 5171–5175.

CE

[27] X. Zhang, H. Zhang, Z. Wu, Z. Wang, H. Niu, C. Li, Nasal absorption enhancement of insulin using PEG-grafted chitosan nanoparticles, Eur J Pharm Biopharm. 68 (3) (2008) 526–534.

AC

[28] X.G. Zhang, D.Y. Teng, Z.M. Wu, X. Wang, Z. Wang, D.M. Yu, C.X. Li, PEG-grafted chitosan nanoparticles as an injectable carrier for sustained protein release, J. Mater. Sci. Mater. Med. vol. 19 (12) (2008) 3525–3533. [29] L. Deng, H. Qi, C. Yao, M. Feng, A. Dong, Investigation on the properties of methoxy poly(ethylene glycol)/chitosan graft co-polymers, J. Biomater. Sci. Polym. Ed. 18 (12) (2007) 1575–1589. [30] Y. Aktas, M. Yemisci, K. Andrieux, R.N. Gursoy, M.J. Alonso, E. Fernandez-Megia et al., Development and brain delivery of chitosan-PEG nanoparticles functionalized with the monoclonal antibody OX26, Bioconjugate Chem. 16 (6) (2005) 1503–1511. 25

ACCEPTED MANUSCRIPT [31] C.A. Falabella, H. Jiang, M.D. Frame, W. Chen, In vivo validation of biological responses of bfgf released from microspheres formulated by blending poly-lactide-co-glycolide and poly(ethylene glycol)-grafted-chitosan in hamster cheek pouch microcirculatory models, Biomater. Sci. Polym. 20 (7-8) (2009) 903–922. [32] K.H. Bae, C.W. Moon, Y. Lee, T.G. Park, Intracellular delivery of heparin complexed with chitosan-g-poly(ethylene glycol) for inducing apoptosis, Pharm Res. 26 (1) (2009)

PT

93–100. [33] G. Ma, X. Zhang, J. Han, G. Song, J. Nie, Photo-polymeriable chitosan derivative prepared

RI

by Michael reaction of chitosan and polyethylene glycol diacrylate (PEGDA), Int J Biol

SC

Macromol. 45 (5) (2009) 499-503.

[34] C.A. Peptu, G. Buhus, M. Popa, A. Perichaud, D. Costin, Double cross-linked chitosan–

NU

gelatin particulate systems for ophthalmic applications, J. Bioact.Compat. Polym. 25 (1) (2010) 98–116.

MA

[35] L. Alupei, C.A. Peptu, A.M. Lungan, J. Desbrieres, O. Chiscan, S. Radji, M. Popa, New hybrid magnetic nanoparticles based on chitosan-maltose derivative for antitumor drug delivery, Int. J. Biol. Macromol. 92 (2016) 561–572.

D

[36] A.N. Jatariu (Cadinoiu), M.N. Holbanb, C.A. Peptu, A. Sava, M. Costuleanu, M. Popa,

PT E

Double crosslinked interpenetrated network in nanoparticle form for drug targeting– Preparation, characterization and biodistribution studies, Int. J. Pharm. 436 (1-2) (2012) 66–74.

CE

[37] P.R. Vuddanda, V.M. Rajamanickam, M. Yaspal and S. Singh, Investigations on Agglomeration and Haemocompatibility of Vitamin E TPGS Surface Modified Berberine

AC

Chloride Nanoparticles, BioMed Research International (2014) 11. [38] J. Chen, Y. Fujino and T. Takahashi, Experimental Uveitis Induced by Intravitreal or Intravenous Lipoteichoic Acid in Rabbits, Jpn. J. Ophthalmol. 43 (5) (1999) 368–374. [39] A. Chatzigeorgiou, A. Halapas, K. Kalafatakis, E. Kamper, The Use of Animal Models in the Study of Diabetes Mellitus, In Vivo 23 (2) (2009) 245–258. [40] W. Chen ,Y. Wu, M. Zheng, Q. Gu, Z. Zheng, X. Xia, Establishing an experimental rat model of photodynamically-induced retinal vein occlusion using erythrosin B, Int. J. Ophthalmol. 7 (2) (2014) 232–238.

26

ACCEPTED MANUSCRIPT [41] S. Rezzola, M. Belleri, G. Gariano, D. Ribatti, C. Costagliola, F. Semeraro, M. Presta, In vitro and ex vivo retina angiogenesis assays, Angiogenesis 17 (3) (2014) 492-442. [42] C. Zang, Q.N. Ping, H. J. Zhang, J. Shen, Synthesis and characterization of water-soluble Osuccinyl-chitosan, Eur. Polym. J. 39 (8) (2003) 1629–1634. [43] C. Peniche, W. A.-Monal, N. Davidenko, R. Sastre, A. Gallardo, J. S. Roman, Self-curing membranes of chitosan/PAA IPNs obtained by radical polymerization: preparation,

PT

characterization and interpolymer complexation, Biomaterials 20 (20) (1999) 1869– 1878.

RI

[44] A.F Martins, J.F. PiaiIvânia, T.A. Schuquel, E.C. Muniz, Polyelectrolyte complexes of chitosan/heparin and N,N,N-trimethyl chitosan/heparin obtained at different pH: I.

SC

Preparation, characterization, and controlled release of heparin, Colloid Polym. Sci. 289, (10) (2011) 1133–1144.

NU

[45] S.P. Facchi, D.B. Scariot, P.V.A. Bueno, P. R. Souza, L.C. Figueiredo, H.D.M. Follmann et al, Preparation and cytotoxicity of N-modified chitosan nanoparticles applied in

MA

curcumin delivery, Int. J. Biol. Macromol. 87 (2016) 237–245. [46] F. Jafary, M. Panjehpour, J. Varshosaz, P. Yaghmaei, Stability improvement of

(2) (2016) 243–250.

D

immobilized alkaline phosphatase using chitosan nanoparticles, Braz. J. Chem. Eng. 33

PT E

[47] Azha. Periasamy, S. Muruganand, M.Palaniswamy, Vibrational studies of Na2SO4, K2SO4, NaHSO4 and KHSO4 crystals, Rasayan J. Chem. 2 (4) (2009), 981–989.

CE

[48] C.A. Contreras, S. Sugita and E. Ramos. Preparation of Sodium Aluminate From Basic Aluminium Sulfate, J. Mater. Process. Adv. Technol. 8 (2) (2006) 122–129. [49] J. Han, J. Zhang, R. Yin, G. Ma, D. Yang, J. Niea, Electrospinning of methoxy

AC

poly(ethylene glycol)-grafted chitosan and poly(ethylene oxide) blend aqueous solution, Carbohydr. Polym. 83 (1) (2011) 270–276. [50] M.A. Dobrovolskaia, J.D. Clogston, B.W. Neun, J.B. Hall, A.K. Patri, S.E. McNeil. Method for Analysis of Nanoparticle Hemolytic Properties, In Vitro Nano Lett. 8 (8) (2008) 2180–2187. [51] D.W. Lee , K. Powers, R. Baney, Physicochemical properties and blood compatibility of acylated chitosan nanoparticles, Carbohydr. Polym. 58 (4) (2004) 371–377.

27

ACCEPTED MANUSCRIPT [52] R. Nadesh, D. Narayanan, P.R. Sreerekha, S. Vadakumpully, U. Mony, M. Koyakkutty, S.V. Nair, D. Menon, Hematotoxicological analysis of surface-modified and -unmodified chitosan nanoparticles, J. Biomed. Mater. Res. Part A 101 (10) (2013) 2957–2966. [53] X. Li, Z. Yang, K. Yang, et al. Self-Assembled Polymeric Micellar Nanoparticles as Nanocarriers for Poorly Soluble Anticancer Drug Ethaselen. Nanoscale Research Lett. 4 (12) (2009) 1502–1511.

PT

[54] P.L. Ritger, N.A. Peppas, A simple equation for description of solute release I. Fickian and non-Fickian release from non-swellable devices in the form of slabs, spheres,

RI

cylinders or discs, J. Control. Release 5 (1) (1987) 23–36.

[55] P.L. Ritger, N.A. Peppas, A simple equation for description of solute release II. Fickian

SC

and anomalous release from swellable devices, J. Control. Release. 5 (1) (1987) 37–42. [56] N.A. Peppas, J.J. Sahlin, A simple equation for the description of solute release. III.

AC

CE

PT E

D

MA

NU

Coupling of diffusion and relaxation, Int. J. Pharm. 57 (2) (1989) 169–172.

28

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Graphical Abstract

29

ACCEPTED MANUSCRIPT

Table captions Table 1. Variation of parameters for the preparation of micro/nanoparticles based on CS-g-

PT

PEGMA crosslinked with sodium tripolyphosphate

Table 2. Variation of parameters for the preparation of micro/nanoparticles CS-g-PEGMA

RI

crosslinked with sodium sulfate

SC

Table 3. Results for thermal analysis in isothermal conditions

Table 5. Zeta potential for optimized MNPs Table 6. Toxicity test averange values

NU

Table 4. Characteristic signals of CS-g-PEGMA and NPs based chitosan derivative

MA

Table 7. Results obtained for the fluorescence of lectin-bound fluorescein for evidence of retinal endothelial cells in control rabbits with Inflammation with lipopolysaccharide, Diabetes and Central Retinal Vein Occlusion in the presence of NPs treatments, whether or not loaded

Figure captions

PT E

D

with the active substance, at 72 hours (%). P <0.05

CE

Scheme 1. The reaction for obtaining chitosan grafted poly(ethylene glycol) methacrylate

AC

Fig. 1. FT-IR spectra of a) PEGMA; b) CS; c) CS-g-PEGMA Fig. 2. 1H NMR spectrum of a) CS; b) PEGMA; c) CS-g-PEGMA Fig. 3. TGA analysis a) CS thermogram b) CS-g-PEGMA thermogram Fig. 4. FT-IR spectra of CS-g-PEGMA and NPs Fig. 5. DSC analysis of CS, CS-g-PEGMA and NPs thermograms Fig. 6. SEM images of MNPs crosslinked with sodium tripolyphosphate Fig. 7. SEM images of MNPs crosslinked with sodium sulphate 30

ACCEPTED MANUSCRIPT Fig. 8. Polymer concentration influence on particle diameter. Granulometric distribution curves of MA-8 (0,5 %), 9 (0,35 %), 11 (0,75 %) crosslinked with TPP Fig. 9. Stirring speed influence on particle diameter. Granulometric distribution curves of MA8 (15.000 rpm), 7 (12.000 rpm), 6(9.000 rpm), 5 (5.000 rpm) crosslinked with sodium tripolyphosphate Fig. 10. Polymers concentration influence on particle diameter. Granulometric distribution

PT

curves of MA-H (0,5 %), I (0,35 %), K (0,75 %) crosslinked with sodium sulphate for Fig. 11. Stirring speed influence on particle diameter. Granulometric distribution curves of

RI

MA-H (15.000 rpm), G (12.000 rpm), F (9.000 rpm), E (5.000 rpm) crosslinked with sodium

SC

sulphate

Fig. 12. Stirring speed influence on swelling process in acidic (ABS)/alkaline (PBS)

NU

environment, [MA-8 and H (15.000 rpm), 7 and G (12.000 rpm), 6 and F (9.000 rpm), 5 and E (5.000 rpm)]

MA

Fig. 13. Polymer concentration influence on swelling process in acidic (ABS)/alkaline (PBS) environment, [MA-9 and I (0,35 %), 8 and H (0,5 %), 11 and K (0,75 %)]

D

Fig. 14. Hemolysis percentage after 2  , 4  and 6  hours exposure to MNPs Fig. 15. Evaluation of osteoblast cell morphology during 5 days in the presence of 5 mg/ml

PT E

MNPs based CS-g-PEGMA

Fig. 16. a) In vitro release kinetics of BEV; b) BEV release efficiency

CE

Fig. 17. Determination of Korsmeyer-Peppas model for BEV release kinetics [MA-8 sample]

AC

Fig. 18. The fluorescein fluorescence (arbitrary units) for retinal endothelial cells for: a) No treatment (100%); b) Inflammation with lipopolysaccharide; c) Diabetes; d) Central Retinal Vein Occlusion

Fig. 19. The fluorescein fluorescence for the 1% suspension of nanoparticle loaded with bevacizumab and retinal endothelial cells (arbitrary units): A) control (100%); B) Inflammation with lipopolysaccharide; C) Diabetes; D) Central Retinal Vein Occlusion Fig. 20. The fluorescein fluorescence for the 1% suspension of nanoparticle loaded with monoclonal antibody (mouse) anti-VEGF-A human (equivalent bevacizumab) and retinal

31

ACCEPTED MANUSCRIPT endothelial cells (arbitrary units): A1) Control (100%); B1) Inflammation with lipopolysaccharide; C1) Diabetes; D1) Central Retinal Vein Occlusion

Tables Table 1. Variation of parameters for the preparation of micro/nanoparticles based on CS-g-

PT

PEGMA crosslinked with sodium tripolyphosphate Polymer

CS/Na5P3O10,

Speed,

Ionic/Covalent

Averange

code

solution

molar ratio

rpm

Cross-linking

Diameter

RI

Sample

concentration, %

duration, h

(SEM), µm

0.5

1:2

5.000

MA-6

0.5

1:2

9.000

MA-7

0.5

1:2

12.000

MA-8

0.5

1:2

15.000

0.50

MA-9

0.35

1:2

15.000

0.50

MA-10

0.5

1:3

15.000

0.80

MA-11

0.75

1:2

SC

MA-5

2.90 1.30

NU

MA 15.000

3.00

1

1.10

Polymer

PT E

crosslinked with sodium sulfate

D

Table 2. Variation of parameters for the preparation of micro/nanoparticles CS-g-PEGMA

Ionic

Covalent

Cross-

Cross-

Averange

Speed,

linking

linking

Diameter

duration, h

duration, h

(SEM), µm

CS/Na2SO4

solution

code

concentration, % ratio

rpm

MA-E

0.5

1:2

5.000

4.40

0.5

1:2

9.000

2.60

0.5

1:2

12.000

1.60

MA-H

0.5

1:2

15.000

1.50

MA-I

0.35

1:2

15.000

1.10

MA-J

0.5

1:3

15.000

1.30

MA-K

0.75

1:2

15.000

MA-G

AC

MA-F

CE

Sample

, molar

1

2

1.10

32

ACCEPTED MANUSCRIPT

Table 3. Results for thermal analysis in isothermal conditions

I

30–100

7.22

II

255–465

45.98

III

465–800

28.25

Calcination

˃ 800

18.54

I

100

II

238–415

III

415–800

8.075

Calcination

˃ 800

2.57

I

PEGMA

II III IV

SC 1.11

NU

88.24

100

5.93

170–308

38.82

350–750

25.54

750–800

8.21

˃ 800

21.5

AC

CE

Calcination

PT

range (0C)

CS-g-

Mass loss, (%)

RI

stages

MA

PEGMA

Temperature

D

Chitosan

Degradation

PT E

Sample

33

ACCEPTED MANUSCRIPT

Table 4. Characteristic signals of CS-g-PEGMA and NPs based chitosan derivative

(cm-1)

Stretching vibrations of -C-O-C

1388, 1643

Signals specific to deformation vibrations of acetylated amine

RI

1072

Absorption band

PT

Wavelength

Compound

groups

The vibration signal corresponding to the C = O bonds

CS-g-

2872

Vibration of CH groups

PEGMA

3436

Axial stretching vibrations of secondary amine groups

1074

Characteristic stretching vibrations -C-O-C

1541

Signals specific to imine linkages -C = N-

1282

Band-specific bonding signal P = O

MA

NU

SC

1722

The absorption band signal corresponding to the new bonds formed

MA-8

by the ionic crosslinking process between polyanions of tripolyphosphate (P-O-P) and ammonium cations of CS-g-PEGMA

1076

Characteristic stretching vibrations -C-O-C

PT E

1548

D

891

Signals specific to imine linkages -C = N-

CE

The absorption band signal corresponding to the bonds formed by

636

SO4 and the CS-g-PEGMA ammonium cations

AC

MA-H

the ionic crosslinking process between the sulfate anions from Na2

Table 5. Zeta potential for optimized MNPs Sample code

Zeta potential [mV]

MA-5

2.275

MA-6

0.543

MA-8

0.583

34

ACCEPTED MANUSCRIPT

Table 6. Toxicity test averange values LD50 –

Hodge-Sterner –

code

average values

toxicity scale

Toxicity level

PT

Sample

(LD50, mg/kg) 500–5000

low toxicity

MA-8

6744

5000–5.000

RI

4922

nontoxic

SC

MA-5

Table 7. Results obtained for the fluorescence of lectin-bound fluorescein for evidence of

NU

retinal endothelial cells in control rabbits with Inflammation with lipopolysaccharide, Diabetes and Central Retinal Vein Occlusion in the presence of NPs treatments, whether or not loaded

MA

with the active substance, at 72 hours (%). P <0.05

Central

D

Inflammation with

No treatment

Control

lipopolysaccharide

Diabetes

Occlusion

100

113.45±4,63

132.74±7.19

120.74±5.12

101.12±3.12

105.77±7.47

117.59±4.91

111.79±3.14

106.19±8.17

120.72±3.79

112.15±3.33

PT E

Applied treatment

Retinal Vein

1% Suspension of BEV Loaded NPs

CE

1% Suspension of

monoclonal antibody

AC

(mouse) anti-VEGF-A human (equivalent bevacizumab) loaded NPs

100.73±2.91

35

ACCEPTED MANUSCRIPT

MA

NU

SC

RI

PT

Figures

Scheme 1. The reaction for obtaining chitosan grafted poly(ethylene glycol)

PT E CE AC

% Transmitance

D

methacrylate

0

500

1732.07 1649.13

a b c

1646.54 1726.21

1000 1500 2000 2500 3000 3500 4000 4500

Wavenumbers, cm

-1

Fig. 1. FT-IR spectra of a) PEGMA; b) CS; c) CS-g-PEGMA 36

ACCEPTED MANUSCRIPT a)

a’

H3 – 6 of acetylated and deacetylated unit

D2O

6 4

5

1

H1 of acetylated and deacetylated unit

PT

3 2 H2

MA

NU

SC

RI

a’

PT E

D

b)

e

f

d

c

AC a

a,b

e

CE

d

c

b

f

37

ACCEPTED MANUSCRIPT c)

PT

H1 of acetylated and deacetylated unit

RI

D2O

PEG backbone and H3 – 6 of acetylated and deacetylated unit

NHA c

NU

SC

CH2bCOO-

AC

CE

PT E

D

MA

Fig. 2. 1H NMR spectrum of a) CS; b) PEGMA; c) CS-g-PEGMA

38

ACCEPTED MANUSCRIPT

b)

AC

CE

PT E

D

MA

NU

SC

RI

PT

a)

Fig. 3. TGA analysis a) CS thermogram b) CS-g-PEGMA thermogram

39

CS-g-PEGMA MA-8 MA-H

1000

1500

2000

2500

3000

NU

500

4000

4500

-1

MA

Wavenumbers, cm

3500

CE

PT E

D

Fig. 4. FT-IR spectra of CS-g-PEGMA and NPs

AC

0

SC

3436.47

RI

2872.00

PT

2322.29

1388.74 1548.98 1643.35 1722.43

891.11 1072.42

636.5

ACCEPTED MANUSCRIPT

Fig. 5. DSC analysis of CS, CS-g-PEGMA and NPs thermograms

40

AC

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 6. SEM images of MNPs crosslinked with sodium tripolyphosphate

41

AC

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 7. SEM images of MNPs crosslinked with sodium sulphate

42

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 8. Polymer concentration influence on particle diameter. Granulometric

AC

CE

PT E

D

MA

distribution curves of MA-8 (0,5 %), 9 (0,35 %), 11 (0,75 %) crosslinked with TPP

Fig. 9. Stirring speed influence on particle diameter. Granulometric distribution curves of MA-8 (15.000 rpm), 7 (12.000 rpm), 6(9.000 rpm), 5 (5.000 rpm) crosslinked with sodium tripolyphosphate

43

SC

RI

PT

ACCEPTED MANUSCRIPT

NU

Fig. 10. Polymers concentration influence on particle diameter. Granulometric distribution curves of MA-H (0,5 %), I (0,35 %), K (0,75 %) crosslinked with sodium

AC

CE

PT E

D

MA

sulphate for

Fig. 11. Stirring speed influence on particle diameter. Granulometric distribution curves of MA-H (15.000 rpm), G (12.000 rpm), F (9.000 rpm), E (5.000 rpm) crosslinked with sodium sulphate

44

ACCEPTED MANUSCRIPT

1000

MA-5 (ABS) MA-5 (PBS) MA-6 (ABS) MA-6 (PBS) MA-7 (ABS) MA-7 (PBS) MA-8 (ABS) MA-8 (PBS)

PT

600

400

RI

Swelling degree, %

800

SC

200

24

NU

0

MA

Time, hours

PT E

MA-E (ABS) MA-E (PBS) MA-F (ABS) MA-F (PBS) MA-G (ABS) MA-G (PBS) MA-H (ABS) MA-H (PBS)

600

CE

Swelling degree, %

800

D

1000

400

AC

200

0 24

Time, hours Fig. 12. Stirring speed influence on swelling process in acidic (ABS)/alkaline (PBS) environment, [MA-8 and H (15.000 rpm), 7 and G (12.000 rpm), 6 and F (9.000 rpm), 5 and E (5.000 rpm)]

45

ACCEPTED MANUSCRIPT 1200

Swelling degree, %

1000

800

600

PT

MA-9 (ABS) MA-9 (PBS) MA-8 (ABS) MA-8 (PBS) MA-11 (ABS) MA-11 (PBS)

400

0 24

NU

Time, hours

MA

1000

D PT E

400

MA-I (ABS) MA-I (PBS) MA-H (ABS) MA-H (PBS) MA-K (ABS) MA-K (PBS)

CE

Swelling degree, %

800

600

200

AC

SC

RI

200

0 24

Time, hours

Fig. 13. Polymer concentration influence on swelling process in acidic (ABS)/alkaline (PBS) environment, [MA-9 and I (0,35 %), 8 and H (0,5 %), 11 and K (0,75 %)]

46

ACCEPTED MANUSCRIPT 1.4

1.2

0.8 0.6

PT

Hemolysis, %

1

0.4

0 100

200

SC

RI

0.2

400

NU

NPs concentration (µg/ml)

MA

Fig. 14. Hemolysis percentage after 2  , 4  and 6  hours exposure to MNPs

MA-6

MA-8

Control sample

AC

CE

PT E

D

MA-5

Fig. 15. Evaluation of osteoblast cell morphology during 5 days in the presence of 5 mg/ml MNPs based CS-g-PEGMA

47

ACCEPTED MANUSCRIPT 0.40 0.35

0.25

PT

0.20 0.15 0.10

RI

mg BEV/mg NPs

0.30

SC

0.05 0.00 0

100

200

300

400

500

a) MA-8 600

NU

Time, hours

MA PT E

D

80

60

20

CE

40

AC

BEV release efficiency , %

100

b) MA-8

0

0

100

200

300

400

500

600

Time, hours Fig. 16. a) In vitro release kinetics of BEV; b) BEV release efficiency

48

ACCEPTED MANUSCRIPT -2 -2.05

0

1

2

3

4

5

y = 0.4968x - 3.8964 R² = 0.9853

-2.1 -2.15 -2.2 -2.25

PT

-2.3 -2.35

RI

-2.4

SC

-2.45

NU

Fig. 17. Determination of Korsmeyer-Peppas model for BEV release kinetics [MA-8 sample]

b

c

d

PT E

D

MA

a

Fig. 18. The fluorescein fluorescence (arbitrary units) for retinal endothelial cells for: a) No

AC

Vein Occlusion

CE

treatment (100%); b) Inflammation with lipopolysaccharide; c) Diabetes; d) Central Retinal

49

ACCEPTED MANUSCRIPT

B

C

D

PT

A

Fig. 19. The fluorescein fluorescence for the 1% suspension of nanoparticle loaded with

RI

bevacizumab and retinal endothelial cells (arbitrary units): A) control (100%); B) Inflammation

NU

SC

with lipopolysaccharide; C) Diabetes; D) Central Retinal Vein Occlusion

B1

C1

D1

PT E

D

MA

A1

Fig. 20. The fluorescein fluorescence for the 1% suspension of nanoparticle loaded with monoclonal antibody (mouse) anti-VEGF-A human (equivalent bevacizumab) and retinal

CE

endothelial cells (arbitrary units): A1) Control (100%); B1) Inflammation with

AC

lipopolysaccharide; C1) Diabetes; D1) Central Retinal Vein Occlusion

50

ACCEPTED MANUSCRIPT Chitosan grafted - poly(ethylene glycol) methacrylate nanoparticles as carrier for controlled release of bevacizumab Highlights

PT

RI

SC NU MA D PT E

•

CE

•

Chitosan (CS) grafted poly(ethylene glycol) methacrylate (CS-g-PEGMA) was synthesized by Michael addition reaction Micro/nanoparticles-based CS-g-PEGMA were prepared via double crosslinking (ionic and covalent) reaction in reverse emulsion Nanoparticles were analyzed from the point of view of their ability to release bevacizumab and the results confirm their potential for the treatment of posterior segment of the eye diseases; in vitro tests showed that the nanoparticles are not cytotoxic and that release bevacizumab from the nanoparticles during several days in a controlled manner.

AC

•

51