Thermoresponsive-co-Biodegradable Linear-Dendritic Nanoparticles

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Thermoresponsive-co-Biodegradable Linear-Dendritic Nanoparticles for Sustained Release of Nerve Growth Factor to Promote Neurite Outgrowth Young Shin Kim, Muhammad Gulfam, and Tao L Lowe Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01044 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Molecular Pharmaceutics

Thermoresponsive-co-Biodegradable Linear-Dendritic Nanoparticles for Sustained Release of Nerve Growth Factor to Promote Neurite Outgrowth

Young Shin Kim†,‡, Muhammad Gulfam§, and Tao L. Lowe†,‡,§,* Departments of Surgery† and Bioengineering‡, Pennsylvania State University, 500 University Drive, Hershey, PA 17033, USA Department of Pharmaceutical Sciences§, University of Tennessee Health Science Center, Memphis, TN 38163, USA

†

Department of Surgery

‡

Department of Bioengineering

§

Department of Pharmaceutical Sciences

* Corresponding Author: E-mail: [email protected] Phone: 901-448-1087

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Molecular Pharmaceutics 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

ABSTRACT: Thermoresponsive and biodegradable linear-dendritic nanoparticles containing poly(Nisopropylacrylamide), poly(L-lactic acid) and poly(L-lysine) dendrons were investigated for sustained release of nerve growth factor (NGF) in response to temperature change. The nanoparticles and their degradants were not cytotoxic to neuron-like PC12 cells for at least one month. The nanoparticles were preferentially taken up by PC12 cells 6 to 13 times more at temperatures above (37 °C) than below (25 °C) the lower critical solution temperature of the nanoparticles. NGF could be loaded into the nanoparticles in aqueous solution and slowly released from the nanoparticles for 12 and 33 days at 25 and 37 °C, respectively. The released NGF was biologically active by promoting neurite outgrowth of PC12 cells. This work demonstrates a new concept of using thermoresponsive and biodegradable linear-dendritic nanoparticles for thermally targeted and sustained release of NGF and other protein drugs for the treatment of Alzheimer’s disease and other neurological disorders.

KEYWORDS: thermoresponsive and biodegradable; linear-dendritic nanoparticles; cellular uptake; nerve growth factor; sustained release; neurite outgrowth

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1. INTRODUCTION Nerve growth factor (NGF) is a member of the neurotrophin family. It is an intracellular signaling protein that enhances the survival, maintenance, and differentiation of cholinergic neurons in the central nervous system, and increases neurite growth and neurotransmitter production.1-3 Therefore, NGF has been proposed as a potential therapy for the treatments of Alzheimer’s disease, stroke and other neurological diseases.4,5 However, NGF does not cross the blood brain barrier, has a short half-life, is easily metabolized when administered peripherally, and possesses biological activity at multiple tissue sites throughout the body.6,7 During the past 15 years, nanoparticles have been exploited for controlled NGF brain delivery, due to their small size, large surface areas and multi-functional surface groups, which characteristics confer good attributes of increasing drug blood circulation time, lowering drug reticuloendothelial system uptake, reducing drug clearance, enhancing drug BBB permeability, and improving drug brain bioavailability.8-21 Linear–dendritic nanoparticles are a type of nanoparticles with intriguing structural properties including a flexible linear polymer chain and densely packed dendron(s) containing internal voids and cavities and a highly branched and functional terminal surface.22-26 The flexible linear polymer chain in the linear–dendritic nanoparticles can offer better loading and slow release for relatively large molecules through chain entanglements than traditional dendrimers with compact structure. The dendritic structure in the linear-dendritic nanoparticles can offer better cell membrane penetration, permeability across biological barriers, and conjugation of targeting moieties and or probes/reporters than the nanoparticles without dendritic structure. Therefore, linear–dendritic nanoparticles have become increasingly interesting for gene therapy25,27-34 and protein delivery.35,36 However, there are no linear–dendritic nanoparticles that have been developed for delivering NGF. Previously we have designed, synthesized, and



characterized proprietary thermoresponsive and biodegradable linear-dendritic nanoparticles containing thermoresponsive poly(N-isopropylacrylamide) (PNIPAAM), hydrophobic and biodegradable poly(L-lactic acid) (PLLA), and cationic and hydrophilic poly(L-lysine) (PLL) dendrons (Figure 1), with a lower critical solution temperature (LCST) in water between 30 and

Thermo-responsive domain PNIPAAM

N H

NH N H N H

L PL n i a om d n dr o O

NH

n De

O

O N H ONH

2

N H

2

O N H O HN

N H 2

C

O

N H N H

2

O

2

HN N H

HN

2

H2N

NH2

2

HN

2

OH OH S O

NH NH O O O O

S O

y

1

x

z -2

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

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HN

H NC O

H O HN O N

O

H N O

H N H N

O

NH2 NH2

NH NH2

O

NH2

O NH2

O

HO

NH2

Biodegradable domain PLLA

Figure 1. Linear-dendritic structure composed of PNIPAAM, PLL, and PLLA polymers that have thermoresponsive and biodegradable properties. U.S. Patent No. 8,916,616 for composition of matter.

37 °C at concentration between 0.05 and 1 mg·mL-1.37 The integration of the PLLA component provides the necessary hydrophobic binding pocket for loading of lipophilic agents via hydrophobic-hydrophobic interactions. PLLA is also degraded by hydrolytic cleavage, which allows for sustained release of bound drug throughout its gradual degradation.38 The incorporation of the thermoresponsive PNIPAAM component offers the advantages of 1) allowing aqueous loading of hydrophilic therapeutic agents at temperatures lower than the LCSTs with high loading efficiency to avoid the use of organic solvents that result in denaturation of therapeutic proteins;39 2) modulating the degradation mechanism of biodegradable polymers;40-42 3) decreasing the cytotoxicity of polycationic polymers;43 4) thermally localizing drugs to targeted sites after systemic injections when their LCSTs are tailored to the temperatures between 37 °C 4 ACS Paragon Plus Environment

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(body temperature) and 42 °C (used routinely in clinical hyperthermia);44-48 and 5) releasing drugs in response to temperature, pH and other stimuli.45,48-50 The inclusion of the cationic and hydrophilic PLL dendrons can enhance delivery of drugs into cells or across biological barriers due to their electrostatic interaction with the polyanionic phospholipids of cell membranes and bulky branched structure.51-53 In the past, we reported the thermoresponsive-co-biodegradable linear-dendritic nanoparticles for sustained release of hydrophobic ceramide to human breast adenocarcinoma cells in response to temperature change.54 In this study, we investigated the utility of the thermoresponsive-co-biodegradable linear-dendritic nanoparticles for aqueous loading of NGF, targeted and sustained delivery of NGF in response to temperature change, and the biological effect of the released NGF on promoting neurite outgrowth in vitro.

2. EXPERIMENTAL SECTION 2.1. Materials The following materials were obtained from Sigma-Aldrich, Inc., St. Louis, MO: Nisopropylacrylamide

(NIPAAM),

1,3-dicyclohexylcarbodiimide

(DCC),

allylamine,

N-

hydroxybenzotriazol (HOBT), 2,5-dihydroxybenzoic acid, N,N-dimethylformamide (DMF, HPLC grade), methylene chloride (CH2Cl2, HPLC grade), ether, methanol, 2-aminoethanethiol, pentanedione peroxide, trifluoroacetic acid (TFA), triisopropylsilane (TIS), piperidine, deuterated chloroform (CDCl3) and tetrahydrofuran (THF), 2,5-dihydroxybenzoic acid, sodium dodecyl sulfate (SDS), fluorescein isothiocyanate (FITC), FITC-labeled dextran (Mw = 4,400 g·mol-1), bovine serum albumin (BSA, Mw = 67 KDa), and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT). Poly(L-lactic acid) (PLLA, Mw = 2,000 g·mol-1 and Mn=1835 g·mol-1) was purchased from Polysciences, Inc., Warrington, PA.


N-alpha-(9-


fluorenylmethyloxycarbonyl)8-Lys4-Lys2-Lys-βAla-Wang

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resin

(Fmoc8-Lys4-Lys2-Lys-βAla-

Wang resin) was purchased from Calbiochem, San Diego, CA. Recombinant human nerve growth factor (NGF, (Mw = 26 kDa) was supplied by Roche Diagnostics (Indianapolis, IN). CellMask™ Deep Red Plasma Membrane Stain and Hoechst 33342 dye were purchased from Thermo Fisher Scientific (Pittsburgh, PA). All of the chemicals were used as received. Deionized distilled water was used in all the experiments. A glass filter frit was purchased from Chemglass, Vineland, NJ. Dialysis membrane (MWCO 3500 Da) was purchased from Spectrum Laboratories (Rancho Dominguez, CA). Proprietary thermoresponsive and biodegradable linear-dendritic nanoparticles containing PNIPAAM, PLLA, and two three-generation branched PLL dendrons (PLL-PNIPAAM grafted with PLLA-PLL, Mw = 5.2 kDa) were synthesized as previously described.37 Briefly, PLLA was acrylated with terminated with allylamine by DCC coupling reaction. The acrylated PLLA was copolymerized with NIPAAM by free radical polymerization. The obtained PNIPAAM grafted with PLLA was conjugated with Fmoc8-Lys4-Lys2-Lys-βAla terminated with carboxylic acid cleaved from Fmoc8-Lys4-Lys2-Lys-βAla-Wang resin by DCC coupling reaction to obtain the final thermoresponsive-co-biodegradable linear-dendritic nanoparticle product. The chemical structure of the nanoparticles was previously characterized/confirmed by 1H nuclear magnetic resonance (NMR), Fourier Transform Infrared Spectroscopy (FTIR) and matrix assisted laser desoption/ionization time of flight (MALDI-TOF).37 The thermoresponsive property of the nanoparticles was previously studied by UV-vis spectroscopy and dynamic light scattering (DLS).37 The results showed that the particle sizes and transmittance of the nanoparticles in water changed with changing the temperature from 10 to 50 °C. The LCSTs of the nanoparticles in water were between 30 and 37 °C at concentrations between 0.05 and 1 mg·mL-1, which


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increased with decreasing the concentration. Below the LCSTs, the nanoparticles were highly swollen with bigger hydrodynamic sizes and 100% transmittance, whereas above the LCSTs, the nanoparticles shrank and collapsed with smaller hydrodynamic sizes and below 95% transmittance. For example, the hydrodynamic diameters of the nanoparticles were 450, 650 and 600 nm at 25 °C, and 125, 250 and 150 nm at 37 °C, at concentrations 0.1, 0.5 and 1 mg·mL-1, respectively.37 In dry state, the diameters of the nanoparticles were between 20-40 nm, measured by transmission electron microscopy (TEM).37 The viscosity and FTIR measurements showed that the nanoparticles hydrolytically degraded for about one month.37

2.2. Cells and cell culture PC12 cells (ATCC, Rockville, MD), a clonal cell line derived from rat adrenal chromaffin cells, were grown in 75 cm2 collagen-coated T-flasks in medium containing 85% RPMI 1640, 5% fetal bovine serum, 10% heat-inactivated horse serum, 3.6 mM L-glutamine, and a penicillin/streptomycin mixture, in a humidified incubator at 37 °C and 5% CO2. Medium components were purchased from GibcoBRL (Grand Island, NY). The cells were harvested every 3-4 days from the tissue culture flasks by brief treatment with trypsin (0.05% trypsin with 0.4 mM EDTA), and split 1:10 at confluence.

2.3. Cell viability PC12 cells were plated onto collagen-coated 96-well plate at a density of 15,000 cells/well (100 µl/well) and the plates were incubated at 37 °C for 1 day. To study the cytotoxicity of the linear-dendritic nanoparticles and their components to PC12 cells, the nanoparticles, PNIAAM alone, PNIPAAM grafted with PLLA, and three-generation branched PLL dendron were added into each well containing PC 12 cells at concentrations of 10, 20, 40, 7 ACS Paragon Plus Environment


80, 150, and 300 µg·mL-1. The plates were incubated at 37 °C for 48 h. Ten microliters of MTT (5 mg/ml in RPMI medium without phenol red) were added to each well. Four hours later, 100 µL of 50% DMF/20% SDS (pH 4.7) mixture solvent was added. The plates were incubated at 37 °C overnight, and then the absorbance at 570 nm was measured using a microplate reader (BioTek Instruments Inc., Winooski, VT) with background subtraction. Cell viability was calculated by dividing the absorbance of the wells containing the nanoparticles/polymers by the absorbance of the wells containing medium only. Each sample and control had four replicates. To study the cytotoxicity of the degradants of the linear-dendritic nanoparticles to PC12 cells, the nanoparticles were dispersed in RPMI medium at 10 mg·ml-1, and allowed to degrade at 37 °C for one month. Weekly, the nanoparticle stock dispersion was diluted into the medium containing PC12 cells at concentrations of 10, 25, 50, 100, 200 and 300 µg·mL-1. The plates were incubated at 37 °C for 48 h and then MTT assay was performed as described above.

2.4. Cellular uptake of linear-dendritic nanoparticles The linear-dendritic nanoparticles were first fluorescently labeled with FITC by reacting amine groups of the linear-dendritic nanoparticles with the thiocyanate group of the FITC. Briefly, FITC in DMF (0.3 mL) and the nanoparticles in bicarbonate buffer (0.1 mol·L-1, 5 mL, pH 9) were mixed at FITC:nanoparticles = 1:16 mole ratio, and the reaction was carried out in the dark at room temperature for 8 h. The final dispersion was dialyzed against distilled water for 10 h, and lyophilized. The percentage of FITC conjugated on nanoparticles was calculated by measuring fluorescence absorbance of the FITC-nanoparticles dispersed in 0.1N NaOH solvent at 494 nm using a microplate reader and a standard curve of FITC in 0.1N NaOH solution. The percentage of FITC conjugated on the nanoparticles was 1.1%. The cellular uptake of the FITC-labeled nanoparticles by PC12 cells at temperatures 8 ACS Paragon Plus Environment

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below (25 °C) and above (37 °C) the LCST of the nanoparticles were studied by confocal microscopy and flow cytometry as follows. PC12 cells were plated at a density of 3.5x104 cells/well onto 12 mm glass coverslips for confocal microscopy experiments and at a density of 4x105 cells/plate into 60 mm tissue culture dishes for flow cytometry experiments. After 1 day, the FITC-labeled nanoparticles at 50, 100, and 200 µg·mL-1 were added into each well/plate and incubated at 25 and 37 °C. Following 1 h treatment, cell culture medium was gently aspirated and the plates were washed twice with 25 or 37 °C PBS (pH 7.4). For confocal microscopy analysis, the cells on the glass coverslips were fixed with 4% paraformaldehyde for 20 min and washed twice with PBS (pH 7.4). Afterwards, the cell membranes were stained with CellMask™ Deep Red Plasma Membrane Stain at 1µg·mL-1 for 10 min, followed by three washings with PBS (pH 7.4). The cellular nuclei were stained with Hoechst 33342 dye at 1µg·mL-1 for 30 min, followed by three final washings with PBS (pH 7.4). The cells were visualized on a Zeiss 710 confocal microscope equipped with seven wavelength (405, 458, 488, 514, 561, 594 and 633 nm) lasers. For flow cytometric analysis, the treated cells were removed by trypsinization, centrifuged (Brinkmann Instruments, NY) at 1,000 rpm, washed with equithermic PBS (pH 7.4), and centrifuged again. The cells were fixed in 4% paraformaldehyde for 20 min and washed twice with PBS (pH 7.4). Each sample was then divided in half, with some samples receiving treatment with 0.5% trypan blue, an extracellular fluorescence-quenching dye, for 5 min followed by two washings with PBS (pH 7.4). This step is necessary in order to differentiate between membrane-bound and internalized linear-dendritic nanoparticles, since trypan blue can not enter living cells. The cell uptake of FITC-labeled nanoparticles was quantitated using a flow cytometric fluorescence-activated cell sorter (FACS, Becton Dickinson, San Jose, CA) equipped with an argon-ion laser and 530 nm bandpass filters for emission measurements. Approximately



10,000 events were acquired per sample, and the data were analyzed using CellQuest software (Becton Dickinson). Forward and side light scatter gates were normally set to exclude dead cells, debris, and cell aggregates. FITC-labeled dextran (Mw = 4,400 g·mol-1) was used as a nonthermoresponsive polymer and positive control.

2.5. NGF loading and release NGF was mixed with the linear-dendritic nanoparticles at a weight ratio of 1:2 in PBS (pH 7.4) and sealed and stored at 4 °C for 3 h. The solution was centrifuged at 39 °C (above the LCST) at 3000 rpm for 1 h. Supernatant was carefully taken out and measured for quantifying NGF loading efficiency. Precipitate was washed with PBS (39 °C) and lyophilized. For NGF release experiment, NGF-loaded nanoparticles were re-suspended in PBS/BSA (pH 7.4, containing 1 w/v% BSA) at 1 mg·mL-1, and stored in a water bath at 25 and 37 °C for 33 days. At selected time points, 10 µL of the dispersion was taken out and centrifuged at 39 °C for 1 h. The supernatant was carefully taken out and mixed with 490 µL of fresh PBS. 100 µL of diluted solution was taken and put into the wells of 96-well plates and stored at 4 °C. NGF amount was quantified by enzyme linked immunosorbent assay (ELISA, Promega, Madison, WI), following the manufacturer’s instructions.

2.6. In vitro biological activities of NGF released from the linear-dendritic nanoparticles PC12 cells were plated onto collagen-coated 48-well at a density of 5,000 cells/well. The plates were incubated at 37 °C for 24 h to allow cells to attach. Ten microliters of NGF-loaded linear-dendritic nanoparticles containing 50 wt% initial NGF loading was added into each well at 10, 50, 150 and 250 µg·mL-1. The plates were incubated at 37 °C for 3 days. The PC12 cells were imaged by inverted microscopy (Nikon ECLIPSE TE2000-5) on Day 1 and 3. The PC12 cell outgrowth was assessed by the number of neurites per cell body, the number of cells having 10 ACS Paragon Plus Environment

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one or more neurites greater than the cell body length, and average neurite length, using NIH ImageJ software. At least 300 cells were examined for each study. The results were normalized with respect to the maximum response and expressed as percentage of clumps bearing neurites. Blank nanoparticles were used as a negative control.

2.7. Statistical Analysis Data were reported as the mean ± standard deviation (SD) from at least three separate experiments. Two-tailed Student’s t-test was used to analyze the differences between treatment groups. A statistically significant difference was reported if p < 0.05.

3. RESULTS 3.1. Cell viability The linear-dendritic nanoparticles and their components PNIPAAM and PNIPAAM grafted with PLLA were not toxic to PC12 cells after 48 h treatment at 37 °C with cell viability higher than 90% at concentration at least up to 300 µg·mL-1, measured by MTT assay (Figure 2a). However, three-generation branched PLL dendron was not toxic to PC12 cells at concentrations equal to or lower than 100 µg·mL-1 with average cell viability ≥90%; but this dendron became slightly toxic to PC12 cells at 200 and 300 µg·mL-1 with cell viability

Thermoresponsive-co-Biodegradable Linear-Dendritic Nanoparticles

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