Low systemic toxicity nanocarriers fabricated from heparin-mPEG and PAMAM dendrimers for controlled drug release

Accepted Manuscript Heparin-mPEG PAMAM dendrimers as low systemic toxicity nanocarriers for controlled drug release

Vu Minh Thanh, Thi Hiep Nguyen, Tuong Vi Tran, Uyen-Thi Phan Ngoc, Minh Nhat Ho, Thi Thinh Nguyen, Yen Nguyen Tram Chau, Le Van Thu, Ngoc Quyen Tran, Cuu Khoa Nguyen, Dai Hai Nguyen PII: DOI: Reference:

S0928-4931(17)31366-8 doi: 10.1016/j.msec.2017.07.051 MSC 8200

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

11 April 2017 28 June 2017 29 July 2017

Please cite this article as: Vu Minh Thanh, Thi Hiep Nguyen, Tuong Vi Tran, UyenThi Phan Ngoc, Minh Nhat Ho, Thi Thinh Nguyen, Yen Nguyen Tram Chau, Le Van Thu, Ngoc Quyen Tran, Cuu Khoa Nguyen, Dai Hai Nguyen , Heparin-mPEG PAMAM dendrimers as low systemic toxicity nanocarriers for controlled drug release, Materials Science & Engineering C (2017), doi: 10.1016/j.msec.2017.07.051

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Heparin-mPEG PAMAM Dendrimers as Low Systemic Toxicity Nanocarriers for Controlled Drug Release Vu Minh Thanh1,2, Thi Hiep Nguyen3, Tuong Vi Tran1,4, Uyen-Thi Phan Ngoc1,4, Minh Nhat Ho1,4, Thi Thinh Nguyen1,4, Yen Nguyen Tram Chau1,4, Le Van Thu5, Ngoc Quyen Tran1,4, Cuu Khoa

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Nguyen1,4, Dai Hai Nguyen3,4,* Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam 2

Tissue Engineering and Regenerative Medicine Group, Department of Biomedical Engineering,

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International University, Vietnam National University-HCMC (VNU-HCMC), HCMC 70000, Vietnam

Institute of Applied Materials Science, Vietnam Academy of Science and Technology

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01 TL29, District 12, Ho Chi Minh City, Vietnam

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Center for High Technology Development, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam

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*Corresponding author: [email protected]

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5

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Institute of Chemistry and Materials, 17 Hoang Sam, Cau Giay, Hanoi, Vietnam.

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Abstract In this report, poly(amide amine) (PAMAM) dendrimer and Heparin-grafted-monomethoxy polyethylene glycol (HEP-mPEG) were synthesized and characterized. In aqueous solution, the generation 4 PAMAM dendrimers (G4.0-PAMAM) existed as nanoparticles with the particle size 5.63 nm. However, after electrostatic complexation with HEP-mPEG to form a core@shell structure G4.0-PAMAM@HEP-mPEG, the size of nanoparticles was significantly increased (73.82 nm). The

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G4.0-PAMAM@HEP-mPEG nanoparticles showed their ability to effectively encapsulate doxorubicin (DOX) for prolonged and controlled release. The cytocompatibility of G4.0PAMAM@HEP-mPEG nanocarriers was significantly increased compared with its parentally G4.0PAMAM dendrimer in both mouse fibroblast NIH3T3 and the human tumor HeLa cell lines. DOX was effectively encapsulated into G4.0-PAMAM@HEP-mPEG nanoparticles to form DOX-loaded

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nanocarriers (DOX/G4.0-PAMAM@HEP-mPEG) with high loading efficiency (73.2%). The release of DOX from DOX/G4.0-PAMAM@HEP-mPEG nanocarriers was controlled and prolonged up to 96 h compared with less than 24 h from their parentally G4.0-PAMAM

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nanocarriers. Importantly, the released DOX retained its bioactivity by inhibiting the proliferation of monolayer-cultured cancer HeLa cells with the same degree of fresh DOX. This prepared G4.0-

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PAMAM@HEP-mPEG nanocarrier can be a potential candidate for drug delivery systems with high loading capacity and low systemic toxicity in cancer therapy.

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Keywords: PAMAM; Heparin; Cancer therapy; Dendrimer; Doxorubicin; Controlled release: Drug

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delivery system

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1. Introduction Poly(amidoamine) (PAMAM) dendrimers, the treelike structure macromolecules, have received enormous attention as potential platforms for drug delivery [1, 2] due to their specific size, shape and chemical functionality. PAMAM are prepared based on a multi-functional ethylene diamine core and an amidoamine repeat branching structure [3]. For the high-generation

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dendrimers, these structures possess internal cavities, which can be used as novel nanocarriers for anticancer drug delivery [4]. Moreover, their externally exposed amine or carboxylic groups that could be decorated with targeting and drug molecules [5]. With the presence of many functional groups on the surface, PAMAM dendrimers are very hydrophilic and highly water-soluble. Therefore, they can be used to covalently attach drugs, targeting ligands or imaging agents for targeted delivery, controlled release, or imaging applications [1, 6]. The potential effect that

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enhanced the drug storage stability, water solubility and anti-tumor elimination of PAMAM were reported when encapsulated with Adriamycin [7], Methotrexate [8] and Paclitaxel [9]. PAMAM can

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enhance the biodistribution of drugs and possibly increase permeation and retention effect for targeting tumors. PAMAM dendrimers have been used to conjugate with different bioactive chemical drugs including Ibuprofen [10], methylprednisolone [11], and N-acetyl cysteine [12] to

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increase the loading capacity, protect drug from denaturation and reduce systematic toxicity due to their capacity to provide targeted drug delivery. The conjugation of PAMAM and Naproxen [13,

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14] showed the PAMAM’s biodistribution is a promising way for drug-resistant cancer [15]. Up to now, PAMAM is still the potential dendrimer for drug resistant cancer due to well delivery drug

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ability and its targeting property.

Despite the extensive interest in the pharmaceutical applications of dendrimers, there is

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conflicting evidence regarding their biological safety that limits its potential clinical application. PAMAM dendrimers have been reported as low biocompatible and high toxicity nanocarriers because of the existence large amount of amino groups on the surface [16, 17]. The strong interaction between the positively charged PAMAM dendrimers and the negatively charged cell membrane may cause disruption to the membrane, hemolytic toxicity and cell lysis [18]. Heparin (HEP), one of the most outstanding negatively charged polymers, has been used to reduce the toxicity of dendrimers by forming the electrostatic interactions with cationic polymers/dendrimers [19-22]. HEP content a relatively high density of sulfate groups which typical provide the anticoagulant and pro-angiogenic characteristics [23, 24]. HEP-based drug delivery systems can provide extra- and intra-cellular interactions to improve the bioactivity of drug and associated

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protein [25-27], and used various anticancer activities in tumor progression and metastatis [28]. Especially, the electrostatic interactions between cationic polymer/dendrimers and anionic HEP are indirectly responsible for the inhibition or enhancement of fibril formation [29]. In addition, HEP has been reported to provide the increase in inner cavity space for improving drug loading content of dendrimer nanocarriers [30-32]. As a result, the electrostatic interaction between HEP and cationic polymers/dendrimers offers a valuable tool for developing drug nanocarriers with minimal

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systemic toxic effect.

Monomethoxy poly(ethylene glycol) (mPEG) has also received great attention owing to its advantages of being coupled with cationic polymers/dendrimers that resulted in reduction of adverse effects from high generation dendrimers [33-36]. The interaction between dendritic copolymers with mPEG leads to water soluble and highly functional hydrid nanomaterials [37].

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mPEG chains reduced and shielded the positive charge on the dendrimer surface and therefore, it can be used for reducing systemic toxicity of the carriers [38-40]. Recently, although the conjugation of PEG into PAMAM could improve a hollow core for drug encapsulation [41] and the

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biocompatibility of the system [39], there is no electrostatic interaction to improve the loading efficiency and prolong the release behavior of the dendrimer nanocarriers. Therefore, to overcome

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these challenges, the surface of PAMAM have been modified with mPEG-conjugated HEP (HEPmPEG) using 4-nitrophenyl chloroformate agent as an intermediate for attaching ethylene glycol to increase the biocompatibility of the system and reduce the cell toxicity [38]. In 2012, Mansoor

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Amiji and co-workers reported a simple new method that modified the surface of polymers with PEG, HEP and albumin for reduced thrombogenicity. The result showed that the thrombogenicity

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was significantly reduced caused by the hydrodynamic properties of HEP and PEG on the surface. This study also demonstrated that the combination between HEP and PEG could be a promising

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candidate for appropriating surface modification of polymeric biomaterials [42]. In this study, generation 4 PAMAM dendrimers (G4.0-PAMAM) and HEP-mPEG were synthesized and employed to fabricate core-shell (G4.0-PAMAM@HEP-mPEG) nanocarriers for doxorubicin (DOX) delivery. The polymer structure and morphology of the obtained nanocarriers were characterized. The cytocompatibility, drug loading efficiency and ability to control the release of loaded drug of the fabricated DOX/G4.0-PAMAM@HEP-mPEG nanocarriers were also evaluated. In addition, the bioactivity of drug released from G4.0-PAMAM@HEP-mPEG nanocarriers was also examined to evaluate their potential application as a drug delivery system. 2. Materials and methods

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2.1 Materials Ethylenediamine (EDA), and methylacrylate (MA) were purchased from Merck (Darmstadt, Germany).

Monomethoxy

polyethylene

glycol

(mPEG,

Mw

5000

Da),

1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide (EDC, 97%), and doxorubicin (DOX) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 4-nitrophenyl chloroformate (PNC) and Heparin (HEP) were purchased from Acros Organics (Geel, Belgium). Dialysis bags, Regenerated Cellulose (MWCO

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3.5 kDa; 6-8 kDa; 12-14 kDa) were purchased from Spectrum Laboratories Inc (CA, USA). All other chemicals were reagent grade and used without further purification.

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2.2 Preparation of PAMAM dendrimers

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Scheme 1. Synthetic route of (a) PAMAM dendrimers and (b) HEP-mPEG PAMAM dendrimers were synthesized using the protocol reported in the literature with minor modification, as shown in Scheme 1a [33, 43]. The synthesis included two main steps: (1) the addition of methylacrylate into the primary amine groups in dendrimer to form the precursor for the next generation, and (2) the amidolization of the coupled ester groups to conjugate primary amine groups to form a completed generation of dendrimers (Scheme 1a) [43]. For example, core precursor, N,N,N’,N’-Tetra(3-methylpropionate), was synthesized using the first step via Michael addition reaction between primary amine groups in EDA and excessive acrylate groups in MA. In detail, MA (40 mL) was dissolved in methanol (40 mL) at 0 oC and then EDA (5.5 mL) was added dropwise via a Pasteur pipette. The reaction mixture was stirred at 0 oC for 3 h and then at room

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temperature (RT) for 2 days, followed by vacuum evaporation to remove methanol and unreacted MA [44]. In the second step, the dendrimer core (G0.0-PAMAM) was synthesized via amidolization the methyl propionate groups in the core precursor using excessive EDA (10-100 times). Specifically, core precursor (20 g) in methanol (10 mL) was slowly added into EDA (130 mL) at 0 oC in 3 h, and the reaction was carried out at RT for 4 days. Excess EDA was later removed by a rotary vacuum evaporator (700-750 mmHg) at 45 oC using toluene/methanol (9/1,

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v/v) mixture, followed by removal of excess toluene using additional methanol for obtaining G0.0PAMAM. The steps 1 (addition) and 2 (amidolization) were alternately repeated to synthesis higher generation PAMAM dendrimers. After each odd step (addition), the generation of PAMAM precursor (e.g., G0.5, G1.5, G2.5, etc.) was obtained with -COOCH3 surface groups while after each even step (amidolization), a completed generation of PAMAM dendrimer (e.g., G1.0, G2.0, G3.0, etc.) was achieved with primary amine groups at the end of branches (Scheme 1a). The rotary

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vacuum evaporator (Strike 300, Lancashire, PR6 0RA, UK) was used to perform the vacuum

round-bottom flask corked tightly [44]. 2.3 Synthesis of HEP-mPEG

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evaporation, and the reaction process was carried out in the dark condition using amber-colored

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mPEG was grafted into HEP using two different steps, including aminolization mPEG to form mPEG-amine and conjugation mPEG-amine into HEP, as shown in Scheme 1b. In detail, mPEG

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(0.16 mmol) was dried at 65 oC under vacuum for 1 h, and PNC (0.19 mmol) was added and the reaction was carried out for 6 h. Thereafter, the reaction mixture was cooled to 40 oC and

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tetrahydrofuran ( 20 mL) was added to dissolve the materials. The obtained solution was slowly dropped into excessive EDA (20 µL) and the obtained mixture was stirred at RT for 24 h, followed by precipitation in excess diethyl ether (500 mL). The mPEG-NH2 was filtered and vacuum-dried at

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RT as yellow-white powder. mPEG-NH2 was then conjugated into HEP using EDC chemistry. Briefly, HEP (0.2 g) and EDC (200 µL) were dissolved in deionized water (diH2O, 15 mL) under constant stirring and mPEG-NH2 (0.12 mmol) was then added into the mixture. After 24 h, the solution was filtered, added into dialysis bags (MWCO 6-8 kDa) and then dialyzed against diH2O, which was changed 5-6 times per day, for 4 days. The obtained solution was lyophilized using a BenchTop "K" Manifold freeze dryer (VirTis, SP Scientific, Warminster, PA, USA) to collect HEPmPEG. The structure of synthesized HEP-mPEG was confirmed using proton NMR. 2.4 Characterizations

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Bruker AC 500 MHz spectrometer (Bruker Co., Billerica, MA, USA) was used to record proton NMR spectroscopy of the sample using D2O as a solvent. The hydrodynamic diameter and zeta potential of nanoparticles were measured at 37 °C using a Zetasizer Nano ZS (ZEN 3600, Malvern Instruments, UK), equipped with a 633 nm wavelength HeNe laser, and the scattered light was detected at 90o. The samples were dispersed in diH2O (1 mg/mL), sonicated for 10 min, filtered (pore size = 0.45 µm) and loaded into the quartz curvet for

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measurement. Zetasizer Nano Series Ver. 5.03 software was used for data acquisition. A transmission electron microscopy (TEM, JEOL 300 kV, JEOL, Japan) was used to observe the morphology of sample solution in diH2O (1 mg/mL) at 37 °C. A drop of the sample solution was placed on a carbon-copper grid (300-mesh, Ted Pella, Inc., USA) and air-dried for 10 min. The samples were stained with 1% (w/v) negatively charged phosphotungstic acid (PTA) for 30 s before

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measurement.

2.5 Preparation of DOX-loaded G4.0-PAMAM@HEP-mPEG nanoparticles (DOX/G4.0-

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PAMAM@HEP-mPEG) and loading efficiency

In this study, the G4.0-PAMAM with theoretically 64 amine groups in the shell was used to prepare G4.0-PAMAM@HEP-mPEG nanoparticles. DOX was loaded in G4.0-PAMAM@HEP-

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mPEG using equilibrium dialysis method as reported earlier [45]. DOX (20 mg) was added into 20 mL G4.0-PAMAM solution in PBS (pH 7.4, 5 mg/mL) with gentle stirring under nitrogen

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atmosphere at RT. The mixture was further gentle stirred overnight followed by adding 200 mg HEP-mPEG in 5 mL PBS and further gentle stirring for 24 h at the same condition. The final

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solution was then dialyzed against diH2O for one day using a dialysis bag (MWCO 3.5 kDa) to remove unloaded DOX from the formulation. The DOX-loaded G4.0-PAMAM@HEP-mPEG

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(DOX/G4.0-PAMAM@HEP-mPEG) solution was then frozen, lyophilized and stored at 4 oC for future experiments. The amount of unloaded DOX (WU-DOX) in dialysis solution was determined using UV-Vis absorbance method. Briefly, fresh DOX was dissolved in diH2O to obtain a series of known concentration solutions (0-60 μg/mL), which were used as standard samples. Absorbance of the standard and dialysis solution samples was recorded at 495 nm using a V-750 UV/Vis spectrophotometer (Jasco Co., Tokyo, Japan). The concentration of DOX in the samples was calculated using a standard curve, which was established based on the relationship between DOX concentrations and absorbance of standard solutions [46, 47]. DOX loading efficiency (LE) was calculated by LE (%) = (20-WU-DOX)/20*100. The DOX loading capacity (LC) was also calculated using the equation, LC (%) = (20-WU-DOX)/(WPAMAM+HEP-mPEG+20-WU-DOX)*100, in which 20 (mg)

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is the initial amount of DOX for the loading experiment, WU-DOX is the total amount of unloaded DOX in the dialysis solution, and WPAMAM+HEP-mPEG is the dried weight of polymer in the nanocarrier. To prepare the empty G4.0-PAMAM@HEP-mPEG nanoparticles, the same procedure was employed except the addition of DOX.

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2.6 In vitro DOX release study DOX release kinetics from G4.0-PAMAM@HEP-mPEG nanocarriers was performed in PBS (10 mM, pH 7.4). 1 mL solution of DOX/G4.0-PAMAM@HEP-mPEG nanocarriers containing 300 g DOX was transferred to dialysis bags (MWCO 12-14 kDa). The sample bags were then immersed into 14 mL fresh release media (PBS pH 7.4) in beakers with constant gentle stirring at RT. At the designated time points, 14 mL of the release medium was collected, and an equal

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volume of fresh medium was added. The amounts of DOX in releases were determined using a UVVis spectrophotometer as mentioned in the previous section.

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2.7 Cell viability assay

The MTT assay was used to evaluate cytotoxicity of synthesized polymers and prepared

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nanocarriers and the bioactivity of released DOX. HeLa cancer cells and normal standard fibroblast cell line NIH3T3 (ATCC, Manassas, VA, USA) were use in this study. The cells were seeded in

medium

(DMEM)

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wells of 96-well plates at a density of 1×104 cells/well in 130 µL of Dulbecco’s Modified Eagle’s supplemented

with

10%

FBS

and

1%

penicillin-streptomycin

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(HyClone, Logan, UT, USA), and cultured for 1 day at 37 oC. Then, the media were removed and the cells were incubated with media containing different sample concentrations for further 48 h, followed by removing media, washing twice with PBS. MTT solution (25 µL, 2 mg/mL) and

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culture medium (130 µL) were added into each well, and the cells were cultured for further 3 h. DMSO (130 µL) was added into each well to dissolve the precipitated purple formazan in 15 min. The samples were then transferred into new transparent 96-wells plates and the absorbance at 570 nm was recorded using a multi-plate reader (SpectraMax M2E, Molecular Devices Co., USA). The cells cultured with medium only were used as a control and assigned to 100% survival. 3. Results and discussion 3.1 Synthesis of PAMAM dendrimers and HEP-mPEG PAMAM dendrimers were synthesized using the protocol reported in the literature with minor modification, as shown in Scheme 1a [33, 43]. Fig. 1a and 1b showed proton NMR with assigned

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protons of G3.5-PAMAM and G4.0-PAMAM. As shown in Fig. 1a, the protons at 2.780-2.846 ppm (peak a), 2.393-2.419 ppm (peak b), 3.268-3.369 ppm (peak c), 2.570-2.634 ppm (peak d) and 3.631-3.689 ppm (peak f) were assigned to protons in methylene groups of =N-CH2-CH2-CO-, =NCH2-CH2-CO-, -CO-NH-CH2-CH2-N=, -CO-NH-CH2-CH2-N= and methyl ended groups –CO-OCH3, respectively. The presence of all these signals demonstrated that G3.5-PAMAM was successfully synthesized and characterized. The additional signals at 3.225-3.259 ppm (g) and

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2.746-2.758 ppm (h) in Fig. 1b were assigned to methylene protons in -CO-NH-CH2-CH2-NH2 and -CO-NH-CH2-CH2-NH2, respectively, of the added ethylenediamine in G4.0-PAMAM. HEP-mPEG was synthesized by grafting mPEG into HEP using two different steps, as shown in Scheme 1b. Fig. 1c shows 1H NMR spectrum of the HEP-mPEG with two hemiacetal proton signals at 5.353 ppm and 5.183 ppm. The signal at 4.580 - 4.648 ppm (peak z) and 3.950-4.071

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(peak k) were assigned to the protons of cyclodextrin ring. The typical peak at 3.751-3.868 ppm (peak l) was assigned to –CH2-CH2–O- groups of mPEG. This result confirmed that mPEG was

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successfully conjugated into HEP [48-50].

Fig. 1. 1H NMR spectra of (a) G3.5-PAMAM, (b) G4.0-PAMAM and (c) HEP-mPEG 3.2 Preparation and characterization of PAMAM@HEP-mPEG nanoparticles In solution, the PAMAM dendrimer exists in the form of core-shell structured nanoparticles, in which the size of G4.0-PAMAM is 5.63 ± 3.83 nm, which was confirmed by DLS data (Fig. 2a,

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dotted curve). After forming electrostatic interaction with HEP in HEP-mPEG, the particle size of formed nanoparticles without and with loaded-DOX, named G4.0-PAMAM@HEP-mPEG and DOX/G4.0-PAMAM@HEP-mPEG, respectively, was significantly increased to 73.82 ± 7.85 nm (Fig. 2a, dashed curve) and 84.25 ± 8.46 nm (Fig. 2a solid curve), respectively. The increase in particle size of G4.0-PAMAM@HEP-mPEG and DOX/G4.0-PAMAM@HEP-mPEG nanoparticles was attributed to the binding of negatively charged HEP-mPEG on to the positively charged surface

charge

of

the

formed

G4.0-PAMAM,

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of PAMAM and the loading of DOX into the nanoparticles. To further investigate the surface G4.0-PAMAM@HEP-mPEG

and

DOX/G4.0-

PAMAM@HEP-mPEG nanoparticles, zeta potential measurement was performed (Fig. 2b). While the zeta potential of cationic G4.0-PAMAM nanoparticles is 23.8 ± 4.71 mV (Fig. 2b, dotted curve), the zeta potential of G4.0-PAMAM@HEP-mPEG dropped down to 2.88 ± 3.14 mV (Fig. 2b, dashed curve) due to the forming of electrostatic interaction with HEP, which decreased the

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density of positively charged group on the surface of the formed nanoparticles. There was a slightly increase in the zeta potential of DOX/G4.0-PAMAM@HEP-mPEG, which is 5.25 ± 4.39 mV (Fig.

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2b solid curve), as compared with G4.0-PAMAM@HEP-mPEG due to the addition of positively

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charged amino groups of DOX molecules.

Fig. 2. (a) Particle size via DLS and (b) zeta potential showing the surface charge of G4.0-PAMAM (dotted curve), G4.0-PAMAM@HEP-mPEG (dashed curve) and DOX/G4.0PAMAM@HEP-mPEG (solid curve) nanoparticles

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In addition, the morphology and particle size of G4.0-PAMAM, DOX-loaded G4.0-PAMAM (DOX/G4.0-PAMAM), G4.0-PAMAM@HEP-mPEG and DOX/G4.0-PAMAM@HEP-mPEG were examined using TEM (Fig. 3). The size of G4.0-PAMAM and DOX/G4.0-PAMAM is under 5 nm and 10 nm in diameter, respectively (Fig. 3a and 3b). However, by complexation with HEP-mPEG, the G4.0-PAMAM@HEP-mPEG and DOX/G4.0-PAMAM@HEP-mPEG possess spherical shape with significant increase in particle size, which is 34.0 ± 17.4 nm and 38.0 ± 13.7 nm for G4.0-

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PAMAM@HEP-mPEG and DOX/G4.0-PAMAM@HEP-mPEG, respectively (Fig. 3c, 3d and e). The increase in particle size of G4.0-PAMAM@HEP-mPEG and DOX/G4.0-PAMAM@HEPmPEG was attributed to the binding of outer layer HEP-mPEG, as shown in Fig. 4. The literature showed that nanoparticles with less than 10 nm in diameter are rapidly cleared from the blood circulation through extravasation [51], whereas the bigger size nanoparticles can retain in circulation at longer period. The advantage of prepared G4.0-PAMAM dendrimers is that the

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bioactive molecules can be loaded in the inner cavity of the dendrimers. To increase the lifetime of the nanoparticles in the blood circulation, the dendrimers could be then formulated with HEP-

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mPEG to increase their size via forming an outer protecting layer, which also help to delay the release rate of loaded bioactive molecules from the nanoparticles in the blood stream. Fig. 4 shows the schematic for loading DOX into core G4.0-PAMAM dendrimer and the complexation of HEP-

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mPEG to form DOX/G4.0-PAMAM@HEP-mPEG nanoparticles, which serves as potential nanocarrier with high drug loading efficiency for long-term circulation and release, and the release

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of DOX from DOX/G4.0-PAMAM@HEP-mPEG nanoparticle over time.

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Fig. 3. TEM images of (a) G4.0-PAMAM, (b) DOX/G4.0-PAMAM, (c) G4.0-PAMAM@HEP-

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mPEG and (d, e) DOX/G4.0-PAMAM@HEP-mPEG

Fig. 4. Schematic showing the loading of DOX to PAMAM dendrimer core, the formation of

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DOX/G4.0-PAMAM@HEP-mPEG nanoparticle and the release of DOX from DOX/G4.0PAMAM@HEP-mPEG over time

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3.3 Cytotoxicity of prepared nanocarriers

In drug delivery system, the cytotoxicity of nanocarriers is an important factor that decides

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their potential applications. The in vitro potential cytotoxicity effect of the synthesized HEP-mPEG, G4.0-PAMAM dendrimer and prepared G4.0-PAMAM@HEP-mPEG nanocarriers against HeLa

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cells and NIH3T3 fibroblast normal healthy cells were then determined (Fig. 5). As shown in Fig. 5a, HEP-mPEG obviously shows cytocompatibility towards both mouse NIH3T3 fibroblast and

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HeLa cancer cells with approximately 90% cell viability at a polymer concentration of 500 µg/mL. In contrast, G4.0-PAMAM cationic dendrimer exhibited the acute cytotoxicity to both cell lines (Fig. 5b). Less than 60% cell viability in both cell lines was achieved at a polymer concentration of

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100 μg/mL and the cell viability dropped to less than 40% at a polymer concentration of 500 μg/mL. Interestingly, the G4.0-PAMAM@HEP-mPEG exhibited the improvement in the cytocompatibility compared with G4.0-PAMAM with more than 80% cell viability for both cell lines at a polymer concentration of 500 μg/mL (Fig. 5c). The improvement in the cytocompatibility of G4.0-PAMAM@HEP-mPEG compared to G4.0-PAMAM can be attributed to the binding of HEP-mPEG at the outer layer of the nanocarriers. The HEP-mPEG outer layer might help to reduce the cytotoxicity of the nanocarrier by decreasing the surface charge and the ability to be internalized through the cell membranes. These results confirm the potential application of G4.0PAMAM@HEP-mPEG as nanocarriers for drug delivery with low systemic toxicity.

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Fig. 5. Viability of monolayer-cultured HeLa cells (triangle) and NIH3T3 fibroblast cells (circle) after 2 days cultured with media containing different concentrations of (a) HEP-mPEG, (b) G4.0-PAMAM and (c) G4.0-PAMAM@HEP-mPEG

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3.4 DOX loading and loading efficiency

The DOX-loaded formulations were prepared using equilibrium dialysis method [45]. The LE

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was calculated indirectly from the amount of unbound DOX, which was determined using spectrophotometrically method. The LE of G4.0-PAMAM and G4.0-PAMAM@HEP-mPEG were 34.7% and 73.2%, respectively. Lower DOX LE in DOX/G4.0-PAMAM was attributed to the

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uncapped surface of the dendrimers, which facilitate the diffusion of DOX over the dialysis process. In contrast, DOX/G4.0-PAMAM@HEP-mPEG possessed a much higher LE because of the adding

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of capped HEP-mPEG molecules on the surface of dendrimer cores, which prevent the diffusion of DOX to the solution. Similarly, DOX/G4.0-PAMAM@HEP-mPEG also possessed a higher LC

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(12.8%) compared to DOX/G4.0-PAMAM (6.5%). These results indicate that introducing of mPEG-HEP resulted in the increasing of both LE and LC of nanoparticles, which may also help to

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increase the ability to control the release of loaded DOX from nanocarriers. 3.5 In vitro DOX release and bioactivity of released DOX In intracellular condition, the release behavior of anticancer drug from nanocarriers is extremely important to not only tumor cells but also healthy tissues. The drug nanocarriers are required to possess high stability, gradual release of loaded drug for long-term courses in the blood circulation [52]. To evaluate the potential application of prepared DOX/G4.0-PAMAM@HEPmPEG nanoparticles for controlled delivery of anticancer drug, in vitro release of DOX from DOX/G4.0-PAMAM

and

DOX/G4.0-PAMAM@HEP-mPEG

was

performed.

DOX/G4.0-

PAMAM@HEP-mPEG nanoparticles showed their potential in controlling the release of DOX for a long-term period up to 96 h (Fig. 6a). After the first 8 h, the cumulative release amount of DOX

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from DOX/G4.0-PAMAM@HEP-mPEG nanoparticles was approximately 29.5% as compared with approximately 79.2% from DOX/G4.0-PAMAM nanoparticles. The initial burst release of DOX from DOX/G4.0-PAMAM@HEP-mPEG nanoparticles could be attributed to the absorbed DOX molecules in the outer HEP-mPEG layers of the nanoparticles. Most of DOX was released from DOX/G4.0-PAMAM nanoparticles within the first day. In contrast, only approximately 74% of loaded DOX was released from DOX/G4.0-PAMAM@HEP-mPEG after 96 h, indicating that G4.0-

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PAMAM@HEP-mPEG nanoparticles served as nanocarriers for stabilizing and controlling the

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release of DOX and/or other anticancer drugs.

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Fig. 6. (a) Cumulative release profiles of DOX from DOX/G4.0-PAMAM and DOX/G4.0PAMAM@HEP-mPEG nanocarriers; (b) bioactivity of released DOX via inhibiting cell

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proliferation: free DOX as a positive control (wide downward diagonal) and released DOX from DOX/G4.0-PAMAM@HEP-mPEG (black)

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To confirm the bioactivity of released DOX from the nanocarriers, the same concentration (1 µg/mL) of free DOX and released DOX from DOX/G4.0-PAMAM@HEP-mPEG in media was cultured with HeLa and NIH3T3 cells for 2 days and their ability to inhibit the cell proliferation was measured using an MTT assay. The cells cultured with medium only were used as a control and assigned to 100% survival. Significant inhibition in cell proliferation was observed in both cell lines when they were treated with the same concentration of free DOX and released DOX from DOX/G4.0-PAMAM@HEP-mPEG (Fig. 6b). More than 50% of HeLa cells were killed when they were treated with free DOX and DOX released from DOX/G4.0-PAMAM@HEP-mPEG nanocarriers as compared with approximately 35% of NIH3T3 fibroblast cells. Importantly, there is no difference in the inhibition degree of cell proliferation between released DOX and fresh DOX

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(Fig. 6b), indicating that the DOX/G4.0-PAMAM@HEP-mPEG nanocarriers could provide the prolonged release of DOX while fully retain the bioactivity of released DOX. Taken together, these results demonstrated that G4.0-PAMAM@HEP-mPEG delivery drug system could be used to control the release of anticancer drug for suppress the growth of cancer cells without significant systemic toxicity.

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4. Conclusion In this study, PAMAM dendrimers and HEP-mPEG were synthesized. G4.0-PAMAM@HEPmPEG nanoparticles have been successfully prepared via electrostatic interaction between cationic PAMAM dendrimer and anionic HEP in HEP-mPEG. The formed G4.0-PAMAM@HEP-mPEG existed in spherical shape with the particle size less than 100 nm and acted as potential nanocarriers for effectively loading and control the release of anticancer drug. The prepared G4.0-

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PAMAM@HEP-mPEG nanocarriers can overcome the cytotoxicity effect of their parentally PAMAM dendrimers and showed high cell viability against both fibroblast NIH3T3 and HeLa

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cancer cell lines at a high polymer concentration. DOX was effectively encapsulated into the formulated G4.0-PAMAM@HEP-mPEG nanoparticles with 73.2% of loading efficiency compared with 29.5 % of G4.0-PAMAM dendrimer. Moreover, the release of DOX from DOX/G4.0-

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PAMAM@HEP-mPEG nanocarriers was prolonged up to 96 h compared with less than 24 h in PAMAM dendrimer. Importantly, the released DOX from DOX/G4.0-PAMAM@HEP-mPEG

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nanocarriers retained its bioactivity by inhibiting the cancer cell proliferation with the same degree of fresh DOX. The developed G4.0-PAMAM@HEP-mPEG nanoparticle could be a promising

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candidate for control the release of anticancer drug with high drug loading efficiency and less systematic cytotoxicity.

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Acknowledgement

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Graphical abstract: Spherical DOX/G4.0-PAMAM@HEP-mPEG nanocarriers with diameter less than 100 nm have been successfully prepared to control and prolong the release of anticancer drug for suppressing the growth of cancer cells without significant systemic toxicity.

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Highlights

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The core@shell structure G4.0-PAMAM@HEP-mPEG have been successfully prepared via electrostatic interaction between cationic PAMAM dendrimer and anionic HEP in H EP-mPEG. The prepared G4.0-PAMAM@HEP-mPEG existed as spherical nanocarriers with diamet er less than 100 nm. The cytocompatibility of G4.0-PAMAM@HEP-mPEG nanocarriers was significantly in creased compared with their parentally G4.0-PAMAM dendrimer in both fibroblast NIH 3T3 and HeLa cell lines. DOX was effectively encapsulated into the G4.0-PAMAM@HEP-mPEG nanoparticles with 73.2% of loading efficiency and its release was prolonged up to 96 h compared wit h only 24 h in PAPAM dendrimer. The released DOX from G4.0-PAMAM@HEP-mPEG nanocarriers retained its bioactivi ty by inhibiting the proliferation of HeLa cells at the same degree of fresh DOX.

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