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Drug-Conjugated Dendrimer Hydrogel Enables Sustained Drug Release via a Self-Cleaving Mechanism Juan Wang, Hongliang He, Remy C. Cooper, Qin Gui, and Hu Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01207 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 13, 2019

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Molecular Pharmaceutics

Drug-Conjugated Dendrimer Hydrogel Enables Sustained Drug Release via a SelfCleaving Mechanism

Juan Wang1, Hongliang He1, Remy C Cooper2, Qin Gui1, Hu Yang1,3,4* 1Department

of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia 23219, United States

2Department

of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia 23284, United States

3Department

of Pharmaceutics, Virginia Commonwealth University, Richmond, Virginia 23298, United States

4Massey

Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298, United States

*To whom correspondence should be addressed: [email protected].

1

ACS Paragon Plus Environment

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 In this study, the anti-cancer drug, camptothecin (CPT), was covalently grafted onto polyamidoamine (PAMAM) dendrimer surface and then reacted with polyethylene glycol diacrylate (PEG-DA) to form dendrimer hydrogel (DH-G3-CPT) with low cross-linking density. In this novel drug delivery system, CPT was cleaved from dendrimer via the ammonolysis of ester bonds and then diffused out of the hydrogel network, thus leading to significantly prolonged drug release. The self-cleaving release kinetics of camptothecin can be further tuned by pH. This DH-G3-CPT drug delivery system has both injectability and sustained drug release. It showed an excellent tumor inhibition effect following intratumoral injection in a head and neck cancer model of mouse. Keywords: dendrimer, hydrogel, self-cleaving, camptothecin, drug delivery

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INTRODUCTION Polyamidoamine (PAMAM) dendrimers have been intensively studied as drug carriers by virtue of their highly branched nanostructures with numerous surface groups and charges.1-7 PAMAM dendrimers can carry drugs in different ways.8-9 Drugs can be either loaded to the dendrimer surface by covalently conjugation or encapsulated into the dendritic interior by hydrophobic interactions.10-18 The way dendrimers are adopted as building units to construct hydrogels not only broadens dendrimer-based drug loading methods but also offers a greater flexibility in formulating drugs.19-28 We have pioneered the use of the highly efficient aza Michael addition to make PAMAM dendrimer hydrogels (DHs) with controllable gelation and degradation kinetics.29-30 In particular, we developed a series of mildly cross-linked DHs for in situ drug loading. 26, 29, 31 The mildly cross-linked DH formulations allow for ease of preparation and are injectable. Nontheless, controlled drug release is not sustained long enough because of the loose network. To preserve injectability, DHs with a mildly cross-linked network need to be maintained. To this end, we shall develop a new drug loading pathway that can enable more sustained drug release from DHs. In this work, we changed the way of loading drug from physically embedding in the gel network to chemically conjugating onto dendrimer building blocks. Camptothecin (CPT) was modified to have an acrylate end group and then grafted onto dendrimer surface. CPT is a clinically used anticancer drug acting to inhibit the activity of topoisomerase I.32 The dendrimer-drug conjugates were then reacted with PEG-DA to form a mildly cross-linked DH (DH-G3-CPT). CPT was firstly cleaved from dendrimer via ammonolysis of the ester bond and then diffuse out of the hydrogel network to be released. This DH drug delivery system with 3



self-cleaving camptothecin has prolonged drug release compared to those physically embedded CPT. DH-G3-CPT tested in a head and neck cancer model showed an excellent tumor inhibition effect following intratumoral injection. MATERIALS AND METHODS Materials. EDA-core PAMAM dendrimer generation 3 (G3) was purchased from Dendritech (Midland, MI). Polyethylene glycol diacrylate (PEG-DA, Mn = 575 g/mol), triethylamine (TEA, 99%), trifluoroacetic acid (TFA), acryloyl chloride, deuterated chloroform (CDCl3), deuterated methanol (MeOD), and WST-1 were purchased from Sigma-Aldrich. (S)(+)-Camptothecin (CPT, 97%) was purchased from AK Scientific, Inc. (Union City, CA). Dichloromethane (DCM), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), and anhydrous magnesium sulfate (MgSO4) were purchased from Fisher Scientific (Pittsburgh, PA). ELISA kits of human IL-1β and IL-6 were purchased from Thermo Fisher Scientific (Waltham, MA). Characterization. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H NMR spectra for structural analysis were obtained on a Bruker 600 MHz spectrometer. High-Performance Liquid Chromatography (HPLC). The purity of CPT, CPT-A, and G3CPT, as well as drug release kinetics, was determined by using an HPLC (Waters) system under the condition described in our previous publication.29 UV−Vis Spectroscopy. The UV-Vis spectra of CPT, CPT-A, and G3-CPT were obtained on an Evolution 201 UV-Visible spectrophotometer (Thermo Scientific). Scanning Electron Microscopy (SEM). The morphologies of dendrimer hydrogel (DH-G3 and DH-G3-CPT) were obtained on a field emission SEM microscope (Hitachi FE-SEM Su4


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70, Japan). Dendrimer gel specimen were lyophilized before sputter-coated with Pt-Au for 90 s. Synthesis of CPT-A. CPT (1 mmol) and TEA (2 mmol) were dissolved in 25 mL of anhydrous DCM, and the mixture was stirred in an ice water bath. Acryloyl chloride (2 mmol, 165 μL) was subsequently added dropwise to the reaction system. The mixture was kept stirring for 2 h and washed with deionized water three times. The DCM phase was dried over MgSO4, filtered, and vacuum dried. The yield of CPT-A was 70%. 1H NMR (600 MHz, CDCl3) δ ppm, 8.43 (d, J = 0.7 Hz), 8.26 (dd, J = 13.3, 5.7 Hz), 7.97 (d, J = 5.5 Hz), 7.86 (dddd, J = 5.6, 4.6, 1.9, 0.9 Hz), 7.70 (ddd, J = 6.2, 3.7, 1.4 Hz), 7.29 (s), 6.60 (dd, J = 11.5, 0.6 Hz), 6.22 (dd, J = 11.5, 7.0 Hz), 6.11 (dd, J = 7.0, 0.6 Hz), 5.77 (dd, J = 19.1, 11.1 Hz), 5.47 (d, J = 11.4 Hz), 5.37-5.32 (m), 2.41-2.19 (m), 1.93 (ddd, J = 14.4, 9.5, 4.7 Hz), 1.06 (dt, J = 18.0, 5.0 Hz). Synthesis of G3-CPT. G3 (0.006 mmol, 43 mg) and CPT-A (0.074 mmol, 30 mg) were dissolved in the DMF/DMSO solvent mixture (6 mL, v/v 1/1) and stirred overnight at room temperature. The reaction mixture was dialyzed against deionized water using dialysis tubing (SnakeSkin, 3.5 K MWCO) and freeze-dried to obtain G3-CPT. Preparation and rheological measurements of dendrimer hydrogels. CPT-conjugated dendrimer hydrogel (DH-G3-CPT) was prepared by mixing 3.2 mg of G3-CPT and 3.0 mg of PEG-DA in 338 μL of PBS followed by gentle shaking overnight. Similarly, plain dendrimer hydrogel DH-G3 was prepared on the basis of 2.1 mg of G3 and 3.0 mg of PEG-DA. To prepare CPT physically loaded dendrimer hydrogel (CPT/DH-G3), 1.1 mg CPT was mixed with 2.1 mg G3 in 338 μL of PBS. After thorough sonication, 3.0 mg of PEG-DA was added to the mixture. CPT/DH-G3 was obtained following overnight shaking. Rheological measurements 5



were performed on a TA Instruments of Discovery hybrid rheometer HR-3 at 25 °C using parallel plate geometry.29 An amplitude sweep was run first to confirm the linear viscoelastic region (LVR). Within LVR, oscillatory frequency sweeps were run at 0.5% strain of in the range of 0.1−100 rad/s. Degradation study of DH-G3. Pre-weighed lyophilized DH-G3 samples (w0) were suspended in PBS (pH 7.4) and incubated at 37 °C. At predetermined time points (6, 24, 48, and 72 h), the residues were collected after centrifugation at 10 000 rpm, washed by deionized water twice, freeze-dried, and weighed (wt). In vitro cytotoxicity assessments. HN12 cancerous cells were seeded at the density of 2 × 104 cells/well in 48-well plate. The cells were cultured for 24 h to allow attachment, and then treated with CPT, G3-CPT, and DH-G3-CPT at different concentrations (equivalent doses 0.5, 5, and 50 μM) for 48 h. Cell viability was determined using the WST-1 assay. To examine cytokines, HN12 cells were seeded in 24-well plate (1×105 cells per well) and cultured 24 h for cell attachment. Upon the removal of the old medium in each well, the cells were incubated in fresh medium for 8 h or 24 h in the presence of CPT, G3-CPT, or DH-G3CPT at an equivalent CPT dose of 0.5 µM. The culture media were collected and centrifuged. The supernatants were subjected to ELISA analysis following manufacturer’s instructions. In vitro drug release kinetics. The release of CPT from various CPT-containing formulations were tested. The formulations included CPT PBS solution, CPT/DH-G3, G3-CPT, and DH-G3-CPT. Each formulation was put in a dialysis tube (0.5k−1k Da MWCO), and the dialysis tube was immersed in 20 mL of PBS equilibrated at 37 °C. At each predetermined time point, 1 mL of release medium was taken and subjected to HPLC analysis for quanitification 6


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of CPT. Immediately following each sampling, 1 mL of fresh PBS was added back. Each release study was repeated three times. In vivo assessment. HN12 head and neck tumor-bearing mouse model was established using male athymic nude mice.33 The tumor-bearing mice were ramdomly divided into three groups (n = 3). Each group received either intratumoral injection of CPT/DH-G3 (160 μg of CPT in 50 μL/mouse), intratumoral injection of DH-G3-CPT (160 μg of CPT in 50 μL/mouse), or nothing. The CPT dose was adopted based on previous publications.34-35 The two treatment groups received one dose at days 10, 13, 16, 20, 22 and 24 post-inoculation of HN12 cells. Tumor volume (Vt = width2 × length/2) and body weight of mice were measured during the experiment. Tumor volume measured at day 10 post-inoculation of tumor cells was set as initial tumor volume (V0). Relative tumor volume (Vt/V0) versus time was plotted to assess tumor response to each treatment. Statistical analysis. The data are reported as means ± standard deviation (SD). One-way analysis of variance (ANOVA) and Student’s t test were conducted for statistical analysis. A p value < 0.05 was considered statistically significant. RESULTS AND DISCUSSION Synthesis and characterization of G3-CPT. G3-CPT (Scheme 1) was synthesized by coupling acrylate modified CPT (CPT-A) to PAMAM G3 via aza-Michael addition. The 1H NMR in Figure 1a showed the chemical shift at 6.60, 6.22, and 6.11 ppm which belong to the unsaturated double bond and thus confirmed the acrylate end-group of CPT-A. According to the proton integrals, each dendrimer was coupled with nine CPT molecules on average (Figure 1b). HPLC analysis (Figure 1c) verified a high purity of both CPT-A and G3-CPT. The eluent 7



time of CPT-A remained the same with CPT (2.55 min). G3-CPT had a shorter eluent time (1.05 min) attributed to the enhanced hydrophilicity after coupling with PAMAM dendrimer. CPT, CPT-A, and G3-CPT exhibited similar spectra with the maximum absorption peak at 365 nm (Figure 1d). Physical property characterization of dendrimer hydrogel. The dendrimer hydrogels (DH-G3 and DH-G3-CPT) were obtained by cross-linking G3 or G3-CPT with PEG-DA at 0.6 wt% (dendrimer) in PBS. The molar ratio of amine/acrylate was kept being constant as 1/1. The two hydrogel formulations remained in liquid form at R.T. but exhibited gel properties as indicated by SEM and rheological results. The SEM images (Figure 2a, b) of both DH-G3 and DH-G3-CPT showed a typical porous gel structure. There were no significant structural differences between DH-G3 and DH-G3-CPT. The pore size of the two gels was around tens of micrometers. The hydrogel DH-G3 was stable within 6 h as there is no mass loss detected (Figure 2c). Nearly 69% of mass remains after 24 h-incubation and the mass dropped to only 7% of after 72 h. An amplitude sweep (Figure 3a, c) was performed first. The LVR was determined to be 0.1%−0.8% for DH-G3 and 0.1%−3% for DH-G3-CPT. The storage modulus (G’) of the two hydrogels were higher than the loss modulus (G”), and their viscoelastic property did not show dependence on frequency in the range of 1 rad/s to ~10 rad/s, the typical hydrogel viscoelastic behavior (Figure 3b, d). Prolonged drug release via self-cleaving ammonolysis. The CPT release profiles of CPT/PBS, CPT/DH-G3, G3-CPT, and DH-G3-CPT are shown in Figure 4. At pH 7.4, the release of physically encapsulated CPT was slightly extended by DH-G3 compared to the PBS formulation. It released 50% of the loaded CPT molecules in 4 h. To understand the physical 8


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loading of CPT by DH-G3, we first measured the solubility of CPT in PAMAM dendrimer G3containing PBS solution (the concentration we used to prepare all the drug-loaded hydrogel formulations). We found that the PBS solution containing 0.6 wt% G3 solubilized 18% more CPT molecules than the PBS solution without G3, hence increasing CPT solubility to 406 µg/mL. The enhanced solubilization of CPT in water was attributed to the entrapment of CPT molecules within the interior of G3. Although a much higher aqueous solubility of CPT could be obtained via dendrimer functionalization,36 not many CPT molecules were entrapped in the dendrimer cores in the current system given the loosely cross-linked network and low dose of DH-G3 used in this study. For the same reason, the release of CPT molecules loaded in the dendrimer hydrogel was not significantly extended. In our previous publications, we have demonstrated the good cell adhesion and cell internalization of the dendrimer gel system.29, 38 In this work, we examined the release of conjugated CPT from G3-CPT and DH-G3-CPT at pH 7.4 and pH 5.3. The pH 5.3 was chosen to mimic an acidic intracellular environment.39 We found that CPT was slowly released from both formulations. We attributed the sustained CPT release to the ammonolysis of ester bonds enabled by those primary amine groups on the periphery of the dendrimer and passive diffusion (Figure 4b). The fact that the rate of ammonolysis of eater bonds is positively correlated to concentration of primary amines40 and a lower pH tends to cause a higher degree of protonation of amines explains why the CPT release rates were higher at pH 7.4 than at pH 5.3 for dendrimer-CPT conjugates. The ammonolysis rate was further decreased in DH-G3-CPT due to the reduced mobility of dendrimers. In vitro cytotoxicity. We tested the cytotoxicity on HN12 cells for three CPT related 9



materials: free CPT, G3-CPT, and DH-G3-CPT. For all the three materials, three different CPT doses (0.5, 5, and 50 µM) were utilized. Figure 5a shows the toxicity results after 48-h incubation. Free CPT can cause 64±6% cell death at 0.5 µM dose, 68±8% cell death at 5 µM dose, and 82±5% cell death at 50 µM dose. G3-CPT and DH-G3-CPT showed much more effective cytotoxicity at 5 µM dose compared to free CPT, leading to 82±4% and 85±6% cell death respectively. Corresponding to the CPT doses of 0.5, 5, and 50 µM, the G3 doses for G3CPT and DH-G3-CPT were 0.055 µM, 0.55 µM, and 5.5 µM. According to literature reports, PAMAM G3 below 10 µM has no toxicity.37 The cytotoxicity of G3-CPT and DH-G3-CPT were all due to the CPT instead of G3. There might be two reasons accounting for the enhanced cytotoxicity of the two materials related with G3-CPT conjugates: 1) After conjugating to dendrimer, the cell internalization of CPT increased. 2) After cell internalization, CPT could be continuously released from G3 and take effect. The conjugation increased the bioavailability of CPT. IL-1β and IL-6 cytokines in the cells were analyzed following treatment with CPT, G3-CPT, and DH-G3-CPT. No IL-1β signal were detected in the experiment. At 8 h, free CPT and G3-CPT increased the IL-6 levels in HN12 cells, whereas DH-G3-CPT reduced the IL-6 level slightly. At 24 h, all the three formulations of CPT, G3-CPT, and DH-G3-CPT increased the IL-6 secretion significantly. The possible reason is that only free CPT and G3-CPT can accelerate IL-6 secretion. It takes time for free CPT and G3-CPT to be cleaved and release from the hydrogel formulation. In vivo tumor inhibition. As shown in Figure 6b, compared to the blank control group, both DH-G3-CPT and CPT/DH-G3 showed tumor inhibition effects. However, DH-G3-CPT showed a significantly more effective tumor inhibition than CPT/DH-G3 after day 16. There 10


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was no obvious body weight loss detected for tumor-bearing mice during the treatment (Figure 6c). Compared to the physically embedded CPT/DH formulation, the chemical conjugation of CPT onto dendrimer has a prolonged duration of drug efficacy. CONCLUSIONS In this work, a camptothecin conjugated dendrimer was adopted to form injectable dendrimer hydrogel. In this drug delivery system, CPT was firstly cleaved from dendrimer via ammonolysis of the ester bond and then released by diffusing out of the hydrogel network. This dendrimer hydrogel with self-cleaving camptothecin has both injectability and prolonged drug release. The formulation showed enhanced in vitro tumor cell toxicity and excellent tumor inhibition effect following intratumoral injection in a xenograft mouse model of head and neck cancer. ACKNOWLEDGMENTS This work was supported, in part, by the National Institutes of Health (R01EY024072).

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core–shell

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A., The influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int J Pharm 2003, 252 (1-2), 263-6. 38.

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Legends Scheme 1. Synthesis of camptothecin-conjugated dendrimer hydrogel. Figure 1. (a) 1H NMR spectrum of CPT-A in CDCl3; (b) 1H NMR spectrum of G3-CPT in MeOD; (c) HPLC chromatograms of CPT, CPT-A, and G3-CPT; (d) UV-Vis spectra of CPT, CPT-A, and G3-CPT. Figure 2. SEM images of (a) DH-G3, (b) DH-G3-CPT, and (c) degradation of DH-G3 at pH 7.4 at 37 °C (n = 3). Figure 3. Oscillatory amplitude sweeps (a, c) and oscillatory frequency sweeps (b, d) of DHG3 (a, b) and DH-G3-CPT (c, d). Figure 4. (a) In vitro drug release profiles at 37 °C (n = 3); (b) The drug release mechanism of DH-G3-CPT. Figure 5. (a) Dose-dependent cytotoxicity of CPT, G3-CPT, and DH-G3-CPT to HN12 cells after 48 h incubation. (b) Cytokine IL-6 expression on HN12 cells. (n = 4). NS indicates not significant and * indicates p < 0.05. Figure 6. In vivo assessment of antitumor effects. (a) Experiment flow; (b) Change of relative tumor volume was monitored throughout treatment; (c) Body weights of mice in all groups were recorded (n = 3). * indicates p < 0.05 and ** indicates p < 0.01.

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N

O

N

O

Et3N

CPT

O

O

O

O

O

O

O N H

H2N 32-x

CPT-A

H N

N

O O

OH O

H2N

NH2 32

N

Cl

N

N

O

O

O

O

O

x

G3-CPT

H N

O

n

O O

O

O

O n

O

NH2

= PAMAM G3 = CPT

N

NH2

N H

O

N

O O

O O

DH-G3-CPT

Scheme 1 19


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

Figure 1 (continued)

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Figure 1

21



Figure 2 22


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Figure 3

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Figure 4 (continued)

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Figure 4

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Figure 5

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Figure 6

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For Table of Contents Use Only

Drug-Conjugated Dendrimer Hydrogel Enables Sustained Drug Release via a SelfCleaving Mechanism Juan Wang1, Hongliang He1, Remy C Cooper2, Qin Gui1, Hu Yang1,3,4*

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Drug-Conjugated Dendrimer Hydrogel Enables ... - ACS Publications

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