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Abstract

The application of hydrogels for anti-cancer drug delivery has garnered considerable interest in the medical field. Current cancer treatment approaches, such as chemotherapy and radiation therapy, often induce severe side effects, causing significant distress and substantial health complications to patients. Hydrogels present an appealing solution as they can be precisely injected into specific sites within the body, facilitating the sustainable release of encapsulated drugs. This localized treatment approach holds great potential for reducing toxicity levels and improving drug delivery efficacy. In this study we developed a hydrogel delivery system containing polyamidoamine (PAMAM) dendrimer and polyethylene glycol (PEG) for solubility enhancement and sustained delivery of hydrophobic anti-cancer drugs. The three selected model drugs, e.g. silibinin, camptothecin, and methotrexate, possess limited aqueous solubility and thus face restricted application. In the presence of vinyl sulfone functionalized PAMAM dendrimer at 45 mg/mL concentration, drug solubility is increased by 37-fold, 4-fold, and 10-fold for silibinin, camptothecin, and methotrexate, respectively. By further crosslinking of the functionalized PAMAM dendrimer and thiolated PEG, we successfully developed a fast-crosslinking hydrogel capable of encapsulating a significant payload of solubilized cancer drugs for sustained release. In water, the drug encapsulated hydrogels release 30%–80% of their loads in 1–4 days. MTT assays of J82 and MCF7 cells with various doses of drug encapsulated hydrogels reveal that cytotoxicity is observed for all three drugs on both J82 and MCF7 cell lines after 48 h. Notably, camptothecin exhibits higher cytotoxicity to both cell lines than silibinin and methotrexate, achieving up to 95% cell death at experimental conditions, despite its lower solubility. Our experiments provide evidence that the PAMAM dendrimer-mediated hydrogel system significantly improves the solubility of hydrophobic drugs and facilitates their sustained release. These findings position the system as a promising platform for controlled delivery of hydrophobic drugs for intratumoral cancer treatment.

Keywords

polyamidoamine dendrimer hydrogel solubility enhancement anti-cancer drugs sustained release

Introduction

Cancer remains a leading cause of mortality in the United States, necessitating the continuous exploration of effective treatment strategies. Currently, chemotherapy and radiation therapy constitute the primary approaches for cancer treatment. However, these therapies are associated with severe side effects and limited delivery efficiency, highlighting the clinical need for enhancing the targeted delivery of therapeutic drugs to cancer sites. In recent years, hydrogels have emerged as promising platforms for drug delivery due to their superior biocompatibility, significant physical properties, and sustained drug release profiles. These advantages position hydrogels favorably over conventional drug delivery approaches which have the drawbacks of systemic toxicity and requirement of repeated dosing.^1–4 Among various kinds of hydrogels for drug delivery, in situ forming hydrogels have gained great interests because they can be delivered to the desired sites through injection, enabling controlled release of the encapsulated drug molecules. The convenience, diversity, and versatility of in situ forming hydrogels have propelled their consideration as intelligent drug delivery systems in various biomedical fields, including tissue regeneration,^5,6 cancer,^7–9 wound healing,^10,11 and antimicrobial or antifungal^12,13 treatments. However, challenges persist in hydrogel-based drug delivery, particularly in solubilizing hydrophobic drug molecules, as the hydrophilic nature of hydrogels may not be inherently compatible with such compounds. Therefore, the development of hydrogel systems capable of enhancing the solubility of hydrophobic drugs is crucial for advancing the technology of hydrogel-based drug delivery in medical fields.

Our research group has developed a series of polyamidoamine (PAMAM) dendrimer-mediated in situ forming hydrogels for tissue engineering.^14–16 PAMAM dendrimers are synthetic star-burst polymers characterized by well-defined structures, non-toxicity, non-immunogenicity, and excellent water solubility.^17–19 These unique properties have earned PAMAM dendrimers fame in a wide range of biomedical applications including imaging, cancer targeting and drug delivery.^19–25 In addition, PAMAM dendrimers have been known for their superb capability to improve the solubility of hydrophobic drugs through electrostatic interactions or hydrogen bonding between surface functional groups of the dendrimer and drugs, or through encapsulation of drug molecules in hydrophobic pockets of their dendritic structures.^26–28 Therefore, PAMAM dendrimer-mediated in situ forming hydrogels could be effective systems for sustained delivery of hydrophobic drugs. In this study, we chose three hydrophobic drug molecules, e.g. silibinin, camptothecin, and methotrexate, whose clinical applications have been hindered due to their low solubility in aqueous solutions. In the presence of vinyl sulfone functionalized PAMAM dendrimer at 45 mg/mL concentration, their aqueous solubility is increased by 37-fold, 4-fold, and 10-fold for silibinin, camptothecin, and methotrexate, respectively. By crosslinking of the same functionalized PAMAM dendrimer and thiolated PEG, fast crosslinking hydrogels are formed with a large payload of solubilized cancer drug encapsulated for extended drug delivery. Release studies reveal that these drug encapsulated hydrogels release 30%–80% of their loads in 1–4 days when immersed in water. MTT assays of J82 and MCF7 cells with different doses of drug encapsulated hydrogels after 48 h demonstrate cytotoxicity for all three drugs. Despite its lower solubility, camptothecin exhibits higher cytotoxicity than silibinin and methotrexate, resulting in up to 95% cell killing at experimental conditions. Collectively, our experiments highlight the remarkable ability of the PAMAM dendrimer-mediated hydrogel system to improve the solubility of hydrophobic drugs and sustain their release, making it a promising platform for controlled delivery of hydrophobic drugs for cancer treatment.

Materials and methods

Polyamidoamine dendrimer generation 5 with terminal amino groups (PAMAM G5-NH₂) is obtained from Dendritech Inc. (Midland, MI, USA). The 8-armed thiolated polyethylene glycol (8arm-PEG-SH, 20 kDa) is purchased from NOF America Corporation (White Plains, NY, USA). Anti-cancer drugs, namely silibinin, camptothecin, and methotrexate (MTX), as well as chemical reagents including acetic anhydride, 2-iminothiolane hydrochloride (2-IT), triethylamine, acrylic acid, divinyl sulfone (DVS), solvents such as dimethyl sulfoxide (DMSO) and methanol are ordered from Fisher Scientific without further purification. MCF7 and J82 cells are acquired from ATCC. Sterilized 48 well plates and MTT kit with inserts are purchased from Fisher Scientific.

Synthesis of PAMAM dendrimer conjugates (dendrimers 2-4)

The vinyl sulfone functionalized PAMAM dendrimer is synthesized in three steps with a procedure reported in our earlier publication.¹⁴ A brief synthetic protocol is provided below. To ensure purity, all dendrimer species are purified with membrane filtration in a stir-cell under pressure followed by thorough washing with phosphate-buffered saline (PBS) buffer and water. The solid products are obtained by lyophilization of the aqueous solution of dendrimer products and characterized by proton nuclear magnetic resonance (¹H NMR) spectrometry.

Synthesis of G5-Ac(70) (dendrimer 2)

PAMAM G5-NH₂ (868 mg, 0.0327 mmol) is dissolved in methanol (100 mL) in a 250 mL round-bottom flask. Triethylamine (290 mg, 2.863 mmol) is added and the reaction mixture is stirred for 5 min. A solution of acetic anhydride (234 mg, 2.290 mmol) in methanol (40 mL) is added dropwise with vigorous stirring. After addition, the resulting mixture is allowed to stir at room temperature overnight. Subsequently, the reaction mixture is transferred into a 600 mL stir-cell equipped with a 10,000 molecular weight cut-off (MWCO) membrane. The residue is extensively washed with PBS buffer (20 mL × 3) and water (20 mL × 3). The aqueous solution of G5-Ac(70) is lyophilized, yielding a solid product (951 mg, 100% yield). ¹H NMR spectrum of G5-Ac(70) is displayed in Figure 1(b). The number of amine groups acetylated is deterimined as 72 by proton intergration of the acetyl methyl group at 1.8 ppm versus the total internal protons in dendrimer, following the method reported.²⁹

Figure 1.

(a) Synthesis of vinyl sulfone functionalized PAMAM dendrimer and drug encapsulated hydrogels. (b)-(d) ¹H NMR spectra in D₂O for dendrimer 2-4.

Synthesis of G5-Ac(70)-SH(40) (dendrimer 3)

G5-Ac(70) (259 mg, 0.00,893 mmol) is dissolved in deionized water (20 mL). A solution of 2-IT (61.4 mg, 0.447 mmol) in deionized water (2 mL) is added dropwise. Nitrogen gas is bubbled into the solution for 2 min to remove the oxygen from media and flask. The reaction mixture is then allowed to stir at room temperature for 2 days. The resulting mixture is transferred to a stir-cell with a 10,000 MWCO membrane and is purified with the same procedure as for dendrimer 2. The product, G5-Ac(70)-SH(40) (276 mg), is obtained as a solid with a yield of 96.6%. ¹H NMR spectrum of G5-Ac(70)-SH(40) is displayed in Figure 1(c). Proton integration of the methylene goups next to thiol at 3.3 and 2.0 ppm versus the acetyl methyl group at 1.8 ppm confirms 42 thiol groups are attached to the dendrimer surface.

Synthesis of G5-Ac(70)-DVS (40) (dendrimer 4)

Dendrimer G5-Ac(70)-SH(40) (155 mg, 0.00,484 mmol) is dissolved in deionized water (20 mL). DVS (114.3 mg, 0.968 mmol) is added to the dendrimer solution. Nitrogen gas is introduced to the mixture for 2 min to remove oxygen. The reaction is allowed to stir at room temperature for 3 days. The resulting mixture is transferred to a stir-cell with a 10,000 MWCO membrane and is purified with the same procedure as for dendrimer 2. The solid product, G5-Ac(70)-DVS(40), is obtained as a powder (164 mg, 96.8% yield). ¹H NMR spectrum of G5-Ac(70)-DVS(40) is displayed in Figure 1(d). The number of DVS attached to the dendrimer is determined as 26 through integration of the alkenyl protons at 6.3 ppm and 6.8 ppm versus the acetyl methyl groups at 1.8 ppm.

Nuclear magnetic resonance (NMR) analysis of PAMAM dendrimers

¹H NMR spectra of dendrimer species are recorded using a Varian 400 MHz spectrometer (Santa Clara, CA, USA) in D₂O solvent.

UV-Vis calibration of cancer drugs

Precisely measure about 2 mg (to 0.00,001 g) of a drug, e.g. silibinin, camptothecin, and methotrexate, and individually dissolve in ethanol (300 μL). Transfer the drug solution to a 100 mL volumetric flask and fill the flask with deionized water to 100 mL. In case the drug is not readily soluble, the solution is gently warmed up to solubilize the drug and then cooled to room temperature while ensuring the drug remains soluble. The resulting drug solution is mixed evenly and diluted to varied concentrations for absorbance measurement using a Shimazu UV-1650PC spectrometer. The absorbances are recorded at the corresponding maximum absorption wavelength for each drug (325 nm for silibinin, 367 nm for camptothecin, and 301 nm for methotrexate). These absorbances are then plotted against the concentrations of the dendrimer to yield a linear calibration curve.

Drug solubility enhancement with PAMAM dendrimers

The solubility enhancement of each drug is evaluated at different dendrimer concentrations. Different aqueous solutions of PAMAM dendrimer 4 with concentrations of 0 mg/mL, 0.938 mg/mL, 1.88 mg/mL, 3.75 mg/mL, 7.50 mg/mL, 15.0 mg/mL, 22.5 mg/mL, 30.0 mg/mL, and 45.0 mg/mL are prepared, each with a volume of 1 mL. To each solution, an appropriate amount of the drug is added, and the mixture is stirred for 1 h. Subsequently, the mixture is centrifuged at 15,000 r/min for 3 minutes to separate the solution from the insoluble residue. The supernatant is collected to measure the drug concentration by UV-Vis spectroscopy. A plot of drug concentrations versus dendrimer concentrations is depicted (Figure 3). As a control, the solubilities of drugs in 8-armed-PEG-SH solutions are also measured using the same procedure and plotted on Figure 3.

Synthesis of PAMAM-PEG hydrogel encapsulated with cancer drugs

Each drug, namely silibinin, camptothecin, and methotrexate, is solubilized in 30 mg/mL dendrimer 4 solution. The mixture is then subjected to centrifugation, and the resulting supernatant is collected for preparation of drug encapsulated hydrogels. The drug concentrations in the supernatant are measured by UV-Vis spectrometry, yielding values of 0.095 mg/mL, 0.034 mg/mL, and 4.65 mg/mL for silibinin, camptothecin, and methotrexate, respectively. To form the drug encapsulated hydrogels, 1 mL of supernatant is mixed in a syringe plunger with a solution containing 8-armed-PEG-SH (47 mg) and water (300 mL). After gelation, cylindrical gels are formed with a diameter of 1.5 cm and a height of 0.7 cm.

UV-Vis measurement of released drugs in PBS

The drug encapsulated hydrogel pellet is immersed in 15 mL distilled water. The concentrations of drug released from hydrogels at 10 min, 30 min, 1 h, 3 h, 8 h, 1 day, 3 days, 5 days, and 7 days are measured with UV-Vis spectroscopy using the calibration curve in Figure 2(a). The drug release profiles are depicted in Figure 4.

MCF7 and J82 cell culture

The MCF7 and J82 cells are obtained from ATCC and cultured in T75 flasks using minimum essential media (MEM from Gibco) supplemented with 10% fetal bovine serum (FBS), and 1% pen-strep. The cells are maintained at 37°C in a CO₂ incubator with a humidified atmosphere of 5% CO₂ and 95% air. The growth media is refreshed every other day. Upon reaching approximately 80% confluency, the MCF7 and J82 cells are harvested from the T75 culture flask for future experiments.

Preparation of anti-cancer drug in hydrogel matrix

The hydrogels are prepared under sterile conditions. The anti-cancer drugs used in this experiment are silibinin, camptothecin, and methotrexate. Stock solutions for drug encapsulated hydrogel formation are as follows. The concentration of PAMAM dendrimer 4 is maintained at 30 mg/mL, while the concentration of PEG solution is set at 47 mg/mL. The concentrations of drugs in PAMAM dendrimer 4 solution (30 mg/mL) are determined to be 0.095 mg/mL, 0.034 mg/mL, and 4.65 mg/mL for silibinin, camptothecin, and methotrexate, respectively. All stock solutions undergo sterile filtration using a 0.22 μm filter. Equal volumes of 8arm-PEG-SH and the respective drug-dendrimer 4 solution are then mixed on the surface of a sterile Petri dish to form a hydrogel. For instance, a hydrogel denoted as 2GD is prepared by mixing 2 μL of 8arm-PEG-SH solution with 2 μL of the drug-PAMAM dendrimer 4 solution. Similarly, samples labeled as 5GD, 10GD, and 30GD are obtained by mixing 5 μL, 10 μL, and 30 μL of 8arm-PEG-SH, respectively, with an equal volume of the drug-PAMAM dendrimer 4 solution. Each hydrogel is prepared in triplet. After gelation, the mixture solidifies into a hydrogel pellet, which is subsequently transferred to the inserts in the 24 well plates.

MTT assays of MCF7 and J82 cells

The viability of MCF7 cells is assessed using the MTT assay. A brief procedure is described as below. MCF7 cells are seeded in four 24 well plates after reaching 80% confluency at a density of 5 × 10⁴ cells and 500 μL fresh MEM media per well. After 2 days of culture, the media in each well is replaced. The drug encapsulated hydrogel pellets prepared earlier are carefully transferred into the insert in each well and are immersed in the media. As a control to each hydrogel with different amount of anti-cancer drugs, an equivalent volume of the drug-dendrimer 4 solution is added in triplet to the same panel. For example, 2PD represents 2 μL drug-dendrimer 4 solution, and the same nomenclature applies to 5PD, 10PD and 30PD. The second control consists of cells without any hydrogel or anti-cancer drugs, indicated as “control” in Figure 5. The third control involves cells treated with hydrogels formed using only 2 μL of 8arm-PEG-SH and 2 μL of PAMAM dendrimer 4 solution without any anti-cancer drugs, denoted as 2G in Figure 5. The cells are then cultured at 37°C in a CO₂ incubator with 5% CO₂ and 95% air. After 48 h of culture, 630 μL of MTT (5 mg/mL in MEM) is added to each well, and the cells are incubated for 3 h at 37°C in a CO₂ incubator. The media is then removed, and 500 μL of PBS buffer is added to wash each well. Subsequently, 250 μL of acidified isopropyl alcohol is added to each well, and the cells are incubated for 5 min. The resulting solution (200 μL) is transferred to a 96 well plates. Triplet wells containing 200 μL of acidified isopropyl alcohol are used as control samples. The absorbance at 570 nm is measured using a Molecular Device microplate reader (SpectraMax M2). The MTT assay for J82 cells follows the same procedure.

Results and discussion

Commercial PAMAM dendrimer G5-NH₂ bearing 110 amino terminal groups is selected for hydrogel preparation. The synthetic scheme is illustrated in Figure 1. The numerical values indicated outside the parentheses of each functional group in the scheme represent the numbers of functional groups that are intended to be attached to dendrimers. These values are not optimized and can be adjusted according to the specific requirement of each application. The synthesis starts with a partial acetylation of PAMAM G5-NH₂ using acetic anhydride, resulting in dendrimer 2, where 70 amino groups are acetylated and 40 amino groups remain available for future chemical modifications. Subsequently, treatment of dendrimer 2 with 2-IT leads to the thiol terminated dendrimer 3. Finally, dendrimer 3 reacts with DVS to accomplish dendrimer 4, which incorporates vinyl sulfone moiety as crosslinkers for hydrogel formation.

The selected model anti-cancer drugs for our study are silibinin, camptothecin, and methotrexate. Silibinin, also known as silybin, has demonstrated significant tumor suppressive activities in a number of cancers by promoting cell apoptosis.³⁰ Camptothecin is a naturally occurring compound with high activity towards several cancer cell lines.³¹ Methotrexate is one of the most frequently used anti-cancer drugs to treat a number of cancers or severe and treatment-resistant autoimmune diseases.³² However, the application of these drugs is restricted by their low water solubility.^31,33,34 Therefore, it is of great importance to develop platforms to deliver those hydrophobic drugs not only in a controllable manner but with enhanced solubility.

In this research, we focus on the development of a PAMAM dendrimer-mediated hydrogel system for delivery of hydrophobic drugs. PAMAM dendrimers are a class of synthetic star-burst polymers that possess well-defined structures as well as desirable properties such as non-toxicity, non-immunogenicity, and excellent water solubility. Their active terminal functional groups allow for chemical modification and bioconjugation to various bioactive molecules, enabling their use in diverse medical applications including cancer targeting and treatment,^35,36 drug delivery,^21,37 gene delivery,^38,39 photodynamic therapy,^40,41 sensors,^42,43 and imaging.^44,45 Furthermore, PAMAM dendrimers have been recognized for their ability to enhance water solubility of hydrophobic drug molecules.^26,27 We propose that a PAMAM dendrimer-mediated hydrogel system will not only increase the solubility of the selected drug molecules but also provide a sustained release for their delivery.

To quantitively determine the amount of drug released, a calibration curve of each drug is to be generated. A range of concentrations of each drug in PBS buffer is prepared, and their UV absorbance is determined on a UV-Vis spectrometer. Figure 2 displays the UV spectra of silibinin, camptothecin, and methotrexate, as well as the corresponding calibration curves. The absorbance measurements are recorded at 325 nm for silibinin, 367 nm for camptothecin, and 301 nm for methotrexate.

Figure 2.

(a) Calibration curves of drugs by UV-Vis (absorbances are measured at 325 nm, 367 nm, and 301 nm for silibinin, camptothecin, and methotrexate, respectively). (b) UV-Vis spectra of silibinin, camptothecin, and methotrexate in aqueous solutions.

To investigate the potential of vinyl sulfone functionalized PAMAM dendrimer (4) in enhancing the aqueous solubility of selected cancer drugs, we individually dissolve silibinin, camptothecin, and methotrexate in solutions containing different concentrations of dendrimer 4, ranging from 0 mg/mL to 45 mg/mL. The aqueous solubility of each drug at each dendrimer concentration is determined using UV-Vis spectrometry. The obtained solubility data for silibinin, camptothecin, and methotrexate are plotted against the concentrations of dendrimer 4, as shown in Figure 3. As a comparison, the solubility of each drug in 8arm-PEG-SH solutions is measured at the same concentrations as dendrimer 4.

Figure 3.

Drug solubility enhancement with dendrimer 4 and PEG concentrations for silibinin (a), camptothecin (b) and methotrexate (c).

It can be seen clearly that the solubility of each drug increases with the concentration of dendrimer 4, while no significant solubility improvement is observed in 8arm-PEG-SH solutions at the same concentrations, demonstrating that PAMAM dendrimer 4 exhibits unique property of solubility enhancement for the hydrophobic drugs tested in this study. Interestingly, the patterns of solubility increase vary for silibinin, camptothecin, and methotrexate. In Figure 3(a), the solubility of silibinin exhibits a remarkable increase as the dendrimer 4 concentrations increase in the experimental range. While in Figure 3(b), the solubility of camptothecin increases with dendrimer 4 concentrations, but the rate of solubility increase slows down at higher dendrimer concentrations. For methotrexate, the solubility increase reaches a plateau at higher dendrimer concentrations, particularly at 30 mg/mL. When dendrimer concentration reaches 45 mg/mL, the solubility of silibinin, camptothecin, and methotrexate has improved by 37-fold, 4-fold, and 10-fold, respectively, compared to the solubility without the addition of dendrimer.

The enhanced solubility of hydrophobic drugs in PAMAM dendrimers is attributed to surface absorption by electrostatic interactions or encapsulation of drugs in dendrimer cavities. Silibinin has been reported to strongly interact with dendrimers through static interactions on surface,⁴⁶ which explains its dramatic improvement in solubility in dendrimer 4 solution. On the other hand, the solubility increase for camptothecin is much lower than that of silibinin in dendrimer 4 solution. This may be attributed to its hydrophobic nature and structural rigidity, which makes it less favorable for encapsulation or interaction with dendrimers. Methotrexate, with higher solubility in water compared to silibinin and camptothecin, shows limited room for solubility improvement in dendrimer solutions before reaching its maximum concentration, which is demonstrated by its higher solubility and its levering off observed in Figure 3(c).

The drug encapsulated hydrogels are prepared according to the scheme in Figure 1. Each drug is saturated in a dendrimer 4 solution at a concentration of 30 mg/mL. After filtering out the insoluble residue, the concentration of each drug in the dendrimer 4 solution is measured by UV-Vis spectroscopy, yielding concentrations of 0.095 mg/mL, 0.034 mg/mL, and 4.65 mg/mL for silibinin, camptothecin, and methotrexate, respectively. Subsequently, 1 mL of the drug-dendrimer 4 solution is mixed with an aqueous solution of 47 mg of 8arm-PEG-SH in 300 μL water in a syringe plunger, with the total numbers of vinyl sulfone groups on dendrimer 4 and thiol groups on PEG roughly in 1:1 ratio. After a gelation time of 10 min to 1 h, a drug encapsulated hydrogel disc is formed with dimensions of 1.5 cm in diameter and 0.7 cm in height. The hydrogel disc is then immersed in 15 mL of deionized (DI) water, and the concentrations of the drug released are measured at different time intervals from 10 min up to 7 days using UV-Vis spectrometry (Figure 4). It is natural that the rate of drug release from hydrogel depends on multiple variables such as the volume, the specific surface area, and the crosslinking density of the gel prepared, the interaction between the drug and gel matrix, and the volume of the medium, etc. Under the experimental conditions, the release profiles for silibinin, camptothecin, and methotrexate exhibit distinct patterns. Silibinin displays more sustained release than the other two drugs as it reaches maximum concentration after 3 days, while camptothecin and methotrexate reach their highest concentrations in 1 day. Figure 4 also displays the percentage of drug release over time. Data show that silibinin, despite its dramatic solubility enhancement by dendrimer 4, only releases 30% of its load, while both camptothecin and methotrexate release 80% of their respective drug loads. The low percentage of release for silibinin might be attributed to the same interactions with PAMAM dendrimer that cause solubility enhancement, which hold silibinin molecules in the hydrogel matrix from release. Conversely, the high percentage of drug release for camptothecin can be attributed to its highly hydrophobic structure and weak interaction with dendrimer once encapsulated in hydrogel. As for methotrexate, unlike camptothecin, the higher drug release may be attributed to its polar nature which facilitates release into the aqueous medium.

Figure 4.

Encapsulated drug release from hydrogels encapsulated with silibinin (a), camptothecin (b) and methotrexate (c).

The cytotoxicity of drug encapsulated hydrogels is evaluated on J82 and MCF7 cell lines using MTT assay. The concentrations of drugs in 30 mg/mL PAMAM stock solutions are 0.095 mg/mL, 0.034 mg/mL, and 4.65 mg/mL for silibinin, camptothecin and methotrexate, respectively. These drug-dendrimer 4 solutions are either added to the cell media at different doses as comparison (the red bars in Figure 5) or mixed with equal volume of 8arm-PEG-SH (47 mg/mL) to form drug encapsulated hydrogels before they are added to the cell media (the blue bars in Figure 5). Experimental data demonstrate that gels produced with dendrimer 4 and 8arm-PEG-SH with no drug encapsulated (the bars labeled with “2G” in all panels) show no cytotoxicity to either J82 or MCF7 cells. However, increased cytotoxicity is observed with the doses of drugs either from the drug-dendrimer 4 stock solution (red bars) or from the drug released from encapsulated gels (blue bars). It is obvious that the drug-dendrimer 4 solutions always exhibit higher cytotoxicity than the drug encapsulated gels except for methotrexate, which shows comparable cytotoxicity for both drug-dendrimer 4 solutions and drug encapsulated gels and on both cell lines. It is worth noticing that camptothecin demonstrates highest cytotoxicity in experimental conditions among the three drugs studied, despite its low solubility in gels (Figure 3). With hydrogel made at the 30 μL camptothecin-dendrimer 4 stock solution (30GD in Figure 5(b) and (e)), the cell killing reaches 95% for both J82 and MCF7 cells, indicating its potency on these cells.

Figure 5.

MTT assay of J82 and MCF7 cells with drug encapsulated hydrogels. The top panels are J82 cells treated with silibinin (a), camptothecin, (b) and methotrexate (c) released from different amounts of hydrogels after 48 h. The bottom panels are MCF7 cells treated with silibinin (d) camptothecin (e) and methotrexate (f) released from different amounts of hydrogels after 48 h. The hydrogels are formed by mixing equal amount of drug-dendrimer 4 solution and 8arm-PEG-SH solution. The controls are cells cultured in media. The numerical numbers in sample labels are the numbers of microliters of solutions used for hydrogel formation. “G” is the notation for gel. “P” is the notation for PAMAM dendrimer 4. “D” is the notation for “drug”. Therefore “2GD” denotes a hydrogel formed with 2 μL of drug-dendrimer 4 solution mixed with 2 μL of 8arm-PEG-SH solution. “2PD” denotes a mixture of 2 μL of drug-dendrimer 4 solution only. “2G” denotes a hydrogel formed by mixing 2 μL of dendrimer 4 and 2 μL of 8arm-PEG-SH solution with no drug added.

Conclusions

A hydrogel system incorporating vinyl sulfone functionalized PAMAM dendrimer and thiolated PEG is developed for solubility enhancement and sustained delivery of hydrophobic anti-cancer drugs. In the presence of functionalized dendrimer 4 at 45 mg/mL concentration, the solubility of silibinin, camptothecin, and methotrexate is increased by 37-folds, 4-folds, and 10-folds, respectively. Subsequently, the PAMAM dendrimer solubilized drug solution is further crosslinked with thiolated PEG to form drug encapsulated hydrogels. These hydrogels exhibit controlled release behavior, with encapsulated drugs released by 30%–80% of their loads in 1–4 days when immersed in water. Cytotoxicity assessments using MTT assays reveal that the drug encapsulated hydrogels exert significant cytotoxicity on both J82 and MCF7 cells, with cell viability reduced by up to 95% after 48 h. With the solubility enhancement and sustained release of the hydrophobic anti-cancer drugs studied, this PAMAM dendrimer-mediated hydrogel system holds promise for intratumoral delivery of hydrophobic drugs in cancer treatment.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Research reported in this publication was supported in part by the NSF and SC EPSCoR/IDeA (MADE in SC) Program under GEAR CRP 20-GC02.

ORCID iD

Xiangdong Bi

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Polyamidoamine dendrimer-mediated hydrogel for solubility enhancement and anti-cancer drug delivery

Abstract

Keywords

Introduction

Materials and methods

Synthesis of PAMAM dendrimer conjugates (dendrimers 2-4)

Synthesis of G5-Ac(70) (dendrimer 2)

Synthesis of G5-Ac(70)-SH(40) (dendrimer 3)

Synthesis of G5-Ac(70)-DVS (40) (dendrimer 4)

Nuclear magnetic resonance (NMR) analysis of PAMAM dendrimers

UV-Vis calibration of cancer drugs

Drug solubility enhancement with PAMAM dendrimers

Synthesis of PAMAM-PEG hydrogel encapsulated with cancer drugs

UV-Vis measurement of released drugs in PBS

MCF7 and J82 cell culture

Preparation of anti-cancer drug in hydrogel matrix

MTT assays of MCF7 and J82 cells

Results and discussion

Conclusions

Footnotes

Declaration of conflicting interests

Funding

ORCID iD

References