Stimuli-responsive hydrogels have enormous potential in drug delivery applications. They can be used for site-specific drug delivery due to environmental variables in the body such as pH and temperature. In this study, we have developed pH-responsive microgels for the delivery of doxorubicin (DOX) in order to optimize its anti-tumor activity while minimizing its systemic toxicity. We used a copolymer of oligo(polyethylene glycol) fumarate (OPF) and sodium methacrylate (SMA) to fabricate the pH-responsive microgels. We demonstrated that the microgels were negatively charged, and the amounts of charge on the microgels were correlated with the SMA concentration in their formulation. The resulting microgels exhibited sensitivity to the pH and ionic strength of the surrounding environment. We demonstrated that DOX was efficiently loaded into the microgels and released in a controlled fashion via an ion-exchange mechanism. Our data revealed that the DOX release was influenced by the pH and ionic strength of the solution. Moreover, we designed a phenomenological mathematical model, based on a stretched exponential function, to quantitatively analyze the cumulative release of DOX. We found a linear correlation between the maximum release of DOX calculated from the model and the SMA concentration in the microgel formulation. The anti-tumor activity of the released DOX was assessed using a human chordoma cell line. Our data revealed that OPF–SMA microgels prolonged the cell killing effect of DOX.
pH-responsive; Doxorubicin; Microgels; Chordoma; Oligo(polyethylene glycol) fumarate
In this study, we have compared the effects of negative and positive fixed charge on chondrocyte behavior in vitro. Electrical charges have been incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) using small charged monomers such as sodium methacrylate (SMA) and (2-(methacryloyloxy) ethyl)-trimethyl ammonium chloride (MAETAC) to produce negatively and positively charged hydrogels, respectively. The hydrogel physical and electrical properties were characterized through measuring and calculating the swelling ratio and zeta potential, respectively. Our results revealed that the properties of these OPF modified hydrogels varied according to the concentration of charged monomers. Zeta potential measurements demonstrated that the electrical property of the OPF hydrogel surfaces changed due to incorporation of SMA and MAETAC and that this change in electrical property was dose-dependent. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy was used to determine the hydrogel surface composition. To assess the effects of surface properties on chondrocyte behavior, primary chondrocytes isolated from rabbit ears were seeded as a monolayer on top of the hydrogels. We demonstrated that the cells remained viable over 7 days and began to proliferate while seeded on top of the hydrogels. Collagen type II staining was positive in all samples; however, the intensity of the stain was higher on negatively charged hydrogels. Similarly, GAG production was significantly higher on negatively charged hydrogels compared to neutral hydrogel. Reverse transcription polymerase chain reaction showed up-regulation of collagen type II and down-regulation of collagen type I on the negatively charged hydrogels. These findings indicate that charge plays an important role in establishing an appropriate environment for chondrocytes and hence in the engineering of cartilage. Thus, further investigation into charged hydrogels for cartilage tissue engineering is merited.
hydrogel; cartilage tissue engineering; OPF; scaffold
An injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) has been investigated as a cell and growth factor carrier for cartilage tissue engineering applications. In this study, hydrogel composites with different swelling ratios were prepared by crosslinking OPF macromers with poly(ethylene glycol) (PEG) repeating units of varying molecular weights from 1,000 ~ 35,000. Rabbit marrow mesenchymal stem cells (MSCs) and MPs loaded with transforming growth factor-β1 (TGF-β1) were encapsulated in the hydrogel composites in order to examine the effect of the swelling ratio of the hydrogel composites on the chondrogenic differentiation of encapsulated rabbit marrow MSCs both in the presence and absence of TGF-β1. The swelling ratio of the hydrogel composites increased as the PEG molecular weight in the OPF macromers increased. Chondrocyte-specific genes were expressed at higher levels in groups containing TGF-β1-loaded MPs and varied with the swelling ratio of the hydrogel composites. OPF hydrogel composites with PEG repeating units of molecular weight 35,000 and 10,000 with TGF-β1-loaded MPs exhibited a 159 ± 95 and a 89 ± 31 fold increase in type II collagen gene expression at day 28, respectively, while OPF hydrogel composites with PEG repeating units of molecular weight 3,000 and 1,000 with TGF-β1-loaded MPs showed a 27 ± 10 and a 17 ± 7 fold increase in type II collagen gene expression, respectively, as compared to the composites with blank MPs at day 0. The results indicate that chondrogenic differentiation of encapsulated rabbit marrow MSCs within OPF hydrogel composites could be affected by their swelling ratio, thus suggesting the potential of OPF composite hydrogels as part of a novel strategy for controlling the differentiation of stem cells.
injectable hydrogels; crosslinking; marrow mesenchymal stem cells; gelatin microparticles; TGF-β1; chondrogenic differentiation; cartilage tissue engineering
In this study, an injectable, biodegradable hydrogel composite of oligo[poly(ethylene glycol) fumarate] (OPF) was investigated as a carrier of mouse embryonic stem cells (mESCs) for the treatment of myocardial infarction (MI). The OPF hydrogels were used to encapsulate mESCs. The cell differentiation in vitro over 14 days was determined via immunohistochemical examination. Then, mESCs encapsulated in OPF hydrogels were injected into the LV wall of a rat MI model. Detailed histological analysis and echocardiography were used to determine the structural and functional consequences after 4 weeks of transplantation. With ascorbic acid induction, mESCs could differentiate into cardiomyocytes and other cell types in all three lineages in the OPF hydrogel. After transplantation, both the 24-hr cell retention and 4-week graft size were significantly greater in the OPF + ESC group than that of the PBS + ESC group (P < 0.01). Four weeks after transplantation, OPF hydrogel alone significantly reduced the infarct size and collagen deposition and improved the cardiac function. The heart function and revascularization improved significantly, while the infarct size and fibrotic area decreased significantly in the OPF + ESC group compared with that of the PBS + ESC, OPF and PBS groups (P < 0.01). All treatments had significantly reduced MMP2 and MMP9 protein levels compared to the PBS control group, and the OPF + ESC group decreased most by Western blotting. Transplanted mESCs expressed cardiovascular markers. This study suggests the potential of a method for heart regeneration involving OPF hydrogels for stem cell encapsulation and transplantation.
cardiac tissue engineering; injectable hydrogels; cell encapsulation; embryonic stem cell; myocardial infarction
In this study, an injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) was investigated as a carrier of mouse embryonic stem cells (mESCs) for the treatment of myocardial infarction (MI). 10K OPF hydrogels were used to encapsulate mESCs. The cell differentiation in vitro over 14 days was determined via immunohistochemical examination. Then, mESCs encapsulated in OPF hydrogels were injected into the left ventricular wall of a rat myocardial infarction model. Detailed histological analysis and echocardiography were used to determine the structural and functional consequences after 4 weeks of transplantation. With ascorbic acid induction, mESCs could differentiate into cardiomyocytes and other cell types in all three lineages in the OPF hydrogel. After transplantation, both the 24h-cell retention and 4-week graft size were significantly greater in the OPF + ESC group than that of the PBS + ESC group (p<0.01). Four weeks after transplantation, OPF hydrogel alone significantly reduced the infarct size and collagen deposition and improved the cardiac function. The heart function and revascularization improved significantly, while the infarct size and fibrotic area decreased significantly in the OPF + ESC group compared with that of the PBS +ESC group, OPF group and PBS group (p<0.01). All treatments had significantly reduced MMP2 and MMP9 protein levels compared to the PBS control group, and the OPF + ESC group decreased most by Western blotting. Transplanted mESCs expressed cardiovascular markers. This study suggest the potential of a method for heart regeneration involving oligo(poly(ethylene glycol) fumarate) hydrogels for stem cell encapsulation and transplantation.
cardiac tissue engineering; injectable hydrogels; cell encapsulation; embryonic stem cell; myocardial infarction
Three-dimensional (3D) tissue-engineered tumor models have the potential to bridge the gap between monolayer cultures and patient-derived xenografts for the testing of nanoparticle (NP)-based cancer therapeutics. In this study, a hydrogel-derived prostate cancer (PCa) model was developed for the in vitro evaluation of doxorubicin (Dox)-loaded polymer NPs (Dox-NPs). The hydrogels were synthesized using chemically modified hyaluronic acid (HA) carrying acrylate groups (HA-AC) or reactive thiols (HA-SH). The crosslinked hydrogel networks exhibited an estimated pore size of 70-100 nm, similar to the spacing of the extracellular matrices (ECM) surrounding tumor tissues. LNCaP PCa cells entrapped in the HA matrices formed distinct tumor-like multicellular aggregates with an average diameter of 50 μm after 7 days of culture. Compared to cells grown on two-dimensional (2D) tissue culture plates, cells from the engineered tumoroids expressed significantly higher levels of multidrug resistance (MDR) proteins, including multidrug resistance protein 1 (MRP1) and lung resistance-related protein (LRP), both at the mRNA and the protein levels. Separately, Dox-NPs with an average diameter of 54 ± 1 nm were prepared from amphiphilic block copolymers based on poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals. Dox-NPs were able to diffuse through the hydrogel matrices, penetrate into the tumoroid and be internalized by LNCaP PCa cells through caveolae-mediated endocytosis and macropinocytosis pathways. Compared to 2D cultures, LNCaP PCa cells cultured as multicellular aggregates in HA hydrogel were more resistant to Dox and Dox-NPs treatments. Moreover, the NP-based Dox formulation could bypass the drug efflux function of MRP1, thereby partially reversing the resistance to free Dox in 3D cultures. Overall, the engineered tumor model has the potential to provide predictable results on the efficacy of NP-based cancer therapeutics.
Hyaluronic acid; hydrogel; 3D tumor model; nanoparticles; cancer therapeutics; drug resistance
We report the development of thermoresponsive
magnetic hydrogels based on poly(N-isopropylacrylamide)
encapsulation of Fe3O4 magnetic nanostructures
(MNS). In particular, we examined the effects of hydrogels encapsulated
with poly-ethylene glycol (PEG) and polyhedral oligomeric silsesquioxane
(POSS) surface modified Fe3O4 MNS on magnetic
resonance (MR) T2 (transverse spin relaxation)
contrast enhancement and associated delivery efficacy of absorbed
therapeutic cargo. The microstructural characterization reveal the
regular spherical shape and size (∼200 nm) of the hydrogels
with elevated hydrophilic to hydrophobic transition temperature (∼40
°C) characterized by LCST (lower critical solution temperature)
due to the presence of encapsulated MNS. The hydrogel-MNS (HGMNS)
system encapsulated with PEG functionalized Fe3O4 of 12 nm size (HGMNS-PEG-12) exhibited relaxivity rate (r2) of 173 mM–1s–1 compared to 129 mM–1s–1 obtained
for hydrogel-MNS system encapsulated with POSS functionalized Fe3O4 (HGMNS-POSS-12) of the same size. Further studies
with HGMNS-PEG-12 with absorbed drug doxorubicin (DOX) reveals approximately
two-fold enhance in release during 1 h RF (radio-frequency) field
exposure followed by 24 h incubation at 37 °C. Quantitatively,
it is 2.1 μg mg–1 (DOX/HGMNS) DOX release
with RF exposure while only 0.9 μg mg–1 release
without RF exposure for the same period of incubation. Such enhanced
release of therapeutic cargo is attributed to micro-environmental
heating in the surroundings of MNS as well as magneto-mechanical vibrations
under high frequency RF inside hydrogels. Similarly, RF-induced in
vitro localized drug delivery studies with HeLa cell lines for HGMNS-PEG-12
resulted in more than 80% cell death with RF field exposures for 1
h. We therefore believe that magnetic hydrogel system has in vivo
theranostic potential given high MR contrast enhancement from encapsulated
MNS and RF-induced localized therapeutic delivery in one nanoconstruct.
poly(N-isopylacrylamide); magneto-thermo responsive polymers; MR active hydrogels; cellular uptake of hydrogels; PEG-functionalized Fe3O4; POSS-functionalized Fe3O4
This work investigated the delivery of marrow mesenchymal stem cells (MSCs), with or without the growth factor transforming growth factor-β1 (TGF-β1), from biodegradable hydrogel composites on the repair of osteochondral defects in a rabbit model. Three formulations of oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel composites containing gelatin microparticles (GMPs) and MSCs were implanted in osteochondral defects, including (1) OPF/GMP hydrogel composites; (2) OPF/GMP hydrogel composites encapsulating MSCs; and (3) OPF hydrogel composites containing TGF-β1 loaded GMPs and MSCs. At 12 weeks, the quality of new tissue formed in chondral and subchondral regions of defects was evaluated based on subjective and quantitative histological analysis. OPF hydrogel composites were partially degraded and the defects were filled with newly formed tissue at 12 weeks with no sign of persistent inflammation. With the implantation of scaffolds alone, newly formed chondral tissue had an appearance of hyaline cartilage with zonal organization and intense staining for glycosaminoglycans, while in the subchondral region hypertrophic cartilage with some extent of bone formation was often observed. The addition of MSCs, especially with TGF-β1 loaded GMPs, facilitated subchondral bone formation, as evidenced by more trabecular bone appearance. However, the delivery of MSCs with or without TGF-β1 at the dosage investigated did not improve cartilage morphology. While OPF-based hydrogel composites supported osteochondral tissue generation, further investigations are necessary to elucidate the effects of MSC seeding density and differentiation stage on new tissue formation and regeneration.
cartilage tissue engineering; mesenchymal stem cells; hydrogel composites; osteochondral defects
We investigated the development of an injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) with encapsulated rabbit marrow mesenchymal stem cells (MSCs) and gelatin microparticles (MPs) loaded with transforming growth factor-β1 (TGF-β1) for cartilage tissue engineering applications. Rabbit MSCs and TGF-β1-loaded MPs were mixed with OPF, a poly(ethylene glycol)-diacrylate crosslinker and the radical initiators ammonium persulfate and N,N,N’,N’-tetramethylethylenediamine, and then crosslinked at 37°C for 8 min to form hydrogel composites. Three studies were conducted over 14 days in order to examine the effects of: 1) the composite formulation, 2) the MSC seeding density, and 3) the TGF-β1 concentration on the chondrogenic differentiation of encapsulated rabbit MSCs. Bioassay results showed no significant difference in DNA amount between groups, however, groups with MPs had a significant increase in glycosaminoglycan content per DNA starting at day 7 as compared to controls at day 0. Chondrocyte-specific gene expression of type II collagen and aggrecan were only evident in groups containing TGF-β1-loaded MPs and varied with TGF-β1 concentration in a dose dependent manner. Specifically, type II collagen gene expression exhibited a 161 ± 49 fold increase and aggrecan gene expression a 221 ± 151 fold increase after 14 days with the highest dose of TGF-β1 (16 ng/ml). These results indicate that encapsulated rabbit MSCs remained viable over the culture period and differentiated into chondrocyte-like cells, thus suggesting the potential of OPF composite hydrogels as part of a novel strategy for localized delivery of stem cells and bioactive molecules.
Cartilage tissue engineering; marrow mesenchymal stem cells; gelatin microparticles; injectable hydrogels; TGF-β1
In this study, a full factorial approach was employed to investigate the effects of poly(ethylene glycol) (PEG) molecular weight (10,000 vs. 35,000 nominal molecular weight), crosslinker-to-macromer carbon-carbon double bond ratio (40 vs. 60), crosslinker type (PEG-diacrylate (PEGDA) vs. N,N′–methylene bisacrylamide (MB)), crosslinking extent of incorporated gelatin microparticles (low vs. high), and incubation medium composition (with or without collagenase) on the swelling and degradation characteristics of oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel composites as indicated by the swelling ratio and the % mass remaining, respectively. Each factor consisted of two levels, which were selected based on previous in vitro and in vivo studies utilizing these hydrogels for various tissue engineering applications. Fractional factorial analyses of the main effects indicated that the mean swelling ratio and the mean % mass remaining of OPF composite hydrogels were significantly affected by every factor. In particular, increasing the PEG chain MW of OPF macromers significantly increased the mean swelling ratio and decreased the mean % mass remaining by 5.7±0.3 and 17.2±0.6 %, respectively. However, changing the crosslinker from MB to PEGDA reduced the mean swelling ratio and increased the mean % mass remaining of OPF composite hydrogels by 4.9±0.2 and 9.4±0.9 %, respectively. Additionally, it was found that the swelling characteristics of hydrogels fabricated with higher PEG chain MW or with MB were more sensitive to increases in DBR. Collectively, the main and cross effects observed between factors enables informed tuning of the swelling and degradation properties of OPF-based hydrogels for various tissue engineering applications.
hydrogels; swelling and degradation; factorial study; poly(ethylene glycol)-based materials; fabrication parameters
Biodegradable oligo(poly(ethylene glycol) fumarate) (OPF) composite hydrogels have been investigated for the delivery of growth factors (GFs) with the aid of gelatin microparticles (GMPs) and stem cell populations for osteochondral tissue regeneration. In this study, a bilayered OPF composite hydrogel that mimics the distinctive hierarchical structure of native osteochondral tissue was utilized to investigate the effect of transforming growth factor-β3 (TGF-β3) with varying release kinetics and/or insulin-like growth factor-1 (IGF-1) on osteochondral tissue regeneration in a rabbit full-thickness osteochondral defect model. The four groups investigated included (i) a blank control (no GFs), (ii) GMP-loaded IGF-1 alone, (iii) GMP-loaded IGF-1 and gel-loaded TGF-β3, and (iv) GMP-loaded IGF-1 and GMP-loaded TGF-β3 in OPF composite hydrogels. The results of an in vitro release study demonstrated that TGF-β3 release kinetics could be modulated by the GF incorporation method. At 12 weeks post-implantation, the quality of tissue repair in both chondral and subchondral layers was analyzed based on quantitative histological scoring. All groups incorporating GFs resulted in a significant improvement in cartilage morphology compared to the control. Single delivery of IGF-1 showed higher scores in subchondral bone morphology as well as chondrocyte and glycosaminoglycan amount in adjacent cartilage tissue when compared to a dual delivery of IGF-1 and TGF-β3, independent of the TGF-β3 release kinetics. The results suggest that although the dual delivery of TGF-β3 and IGF-1 may not synergistically enhance the quality of engineered tissue, the delivery of IGF-1 alone from bilayered composite hydrogels positively affects osteochondral tissue repair and holds promise for osteochondral tissue engineering applications.
Hydrogel; osteochondral defect; transforming growth factor-β3; insulin-like growth factor-1
Oligo (polyethylene glycol) fumarate (OPF) hydrogel has been employed in musculoskeletal tissue engineering for photo-encapsulation of chondrocytes and as a matrix for marrow stromal cells differentiation. In this study, we have studied the application of OPF hydrogel for co-encapsulation of DNA and bone cells and examined whether co-encapsulation can enhance gene transfer by maintaining the DNA within the cellular microenvironment. Our results showed that plasmid DNA encoding green fluorescence protein (GFP), co-encapsulated with bone tumor cells, was capable of transfecting the cells and the transfected tumor cells continuously expressed GFP protein over the time course of study (21 days). Furthermore, we have examined the co-encapsulation of estrogen receptor (ER) encoding plasmid DNA and human fetal osteoblast cells (hFOB) that lack endogenous ER. Our results show that the transfected cells responded to estrogen as alkaline phosphatase (ALP), and estrogen response element (ERE)-directed luciferase enzyme activities increased with estrogen-treatment. Taken together, these studies show that OPF hydrogel could be further explored for targeted gene delivery in bone and other tissues encapsulated within the hydrogels.
Bone tissue engineering; DNA delivery, Hydrogel; Osteoblast, Estrogen receptor
The transected rat thoracic (T9/10) spinal cord model is a platform for quantitatively compa0ring biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ε-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF+ hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF+ polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.
OPF; PLGA; PCLF; axon regeneration; spinal cord injury; Schwann cell
An exciting approach to tumor delivery is encapsulation of the drug in self-assembled polymer-peptide nanoparticles. The objective of this work was to synthesize a conjugate of low molecular weight polylactide (LMW PLA) and V6K2 peptide, and investigate self-assembly, drug release kinetics, cell uptake and toxicity, drug pharmacokinetics, and tumor cell invasion with Doxorubicin (DOX) or paclitaxel (PTX). The results for PLA-V6K2 self-assembled NPs were compared with those of polyethylene glycol stabilized PLA (PLA-EG) NPs. The size of PLA-V6K2 and PLA-EG NPs were 100±20 and 130±50 nm, respectively, with polydispersity index of 1.04 and 1.14. The encapsulation efficiency of DOX in PLA-V6K2 and PLA-EG NPs was 44±9% and 55±5%, respectively, and that of PTX was >90 for both NP types. The release of DOX and PTX from PLA-V6K2 was slower than that of PLA-EG and the release rate was relatively constant with time. Based on molecular dynamic simulation, the less hydrophobic DOX was distributed in the lactide core as well as the peptide shell while the hydrophobic PTX was localized mainly to the lactide core. PLA-V6K2 NPs had significantly higher cell uptake by 4T1 mouse breast carcinoma cells compared to PLA-EG NPs, which was attributed to the electrostatic interactions between the peptide and negatively charged moieties on the cell membrane. PLA-V6K2 NPs showed no toxicity to marrow stromal cells. DOX loaded PLA-V6K2 NPs showed higher toxicity to 4T1 cells and the DNA damage response and apoptosis was delayed compared to the free DOX. DOX or PTX encapsulated in PLA-V6K2 NPs significantly reduced invasion of 4T1 cells compared to those cells treated with the drug in PLA-EG NPs. Invasion of 4T1 cells treated with DOX in PLA-V6K2 and PLA-EG NPs was 5±1% and 30±5%, respectively, and that of PTX was 11±2% and 40±7%. The AUC of DOX in PLA-V6K2 NPs was 67% and 21% higher than those of free DOX and PLA-EG NPs, respectively. DOX loaded PLA-V6K2 NPs injected in C3HeB/FeJ mice inoculated with MTCL syngeneic breast cancer cells displayed higher tumor toxicity than PLA-EG NPs and lower host toxicity than the free DOX. Cationic PLA-V6K2 NPs with higher tumor toxicity than the PLA-EG NPs are potentially useful in chemotherapy.
Self-assembling peptide; polymer conjugation; hybrid nanoparticle; cell uptake; drug pharmacokinetics; tumor toxicity
Management of osteochondritis dissecans remains a challenge. Use of oligo[poly(ethylene glycol)fumarate] (OPF) hydrogel scaffold alone has been reported in osteochondral defect repair in small animal models. However, preclinical evaluation of usage of this scaffold alone as a treatment strategy is limited.
We therefore (1) determined in vitro pore size and mechanical stiffness of freeze-dried and rehydrated freeze-dried OPF hydrogels, respectively; (2) assessed in vivo gross defect filling percentage and histologic findings in defects implanted with rehydrated freeze-dried hydrogels for 2 and 4 months in a porcine model; (3) analyzed highly magnified histologic sections for different types of cartilage repair tissues, subchondral bone, and scaffold; and (4) assessed neotissue filling percentage, cartilage phenotype, and Wakitani scores.
We measured pore size of freeze-dried OPF hydrogel scaffolds and mechanical stiffness of fresh and rehydrated forms. Twenty-four osteochondral defects from 12 eight-month-old micropigs were equally divided into scaffold and control (no scaffold) groups. Gross and histologic examination, one-way ANOVA, and one-way Mann-Whitney U test were performed at 2 and 4 months postoperatively.
Pore sizes ranged from 20 to 433 μm in diameter. Rehydrated freeze-dried scaffolds had mechanical stiffness of 1 MPa. The scaffold itself increased percentage of neotissue filling at both 2 and 4 months to 58% and 54%, respectively, with hyaline cartilage making up 39% of neotissue at 4 months.
Rehydrated freeze-dried OPF hydrogel can enhance formation of hyaline-fibrocartilaginous mixed repair tissue of osteochondral defects in a porcine model.
Rehydrated freeze-dried OPF hydrogel alone implanted into cartilage defects is insufficient to generate a homogeneously hyaline cartilage repair tissue, but its spacer effect can be enhanced by other tissue-regenerating mediators.
Nanoscale polymeric micelles have attracted more and more attention as a promising nanocarrier for controlled delivery of antineoplastic drugs. Herein, the doxorubicin (DOX)-loaded poly(D-lactide)-based micelle (PDM/DOX), poly(L-lactide)-based micelle (PLM/DOX), and stereocomplex micelle (SCM/DOX) from the equimolar mixture of the enantiomeric four-armed poly(ethylene glycol)-polylactide (PEG-PLA) copolymers were successfully fabricated. In phosphate-buffered saline (PBS) at pH 7.4, SCM/DOX exhibited the smallest hydrodynamic diameter (Dh) of 90 ± 4.2 nm and the slowest DOX release compared with PDM/DOX and PLM/DOX. Moreover, PDM/DOX, PLM/DOX, and SCM/DOX exhibited almost stable Dhs of around 115, 105, and 90 nm at above normal physiological condition, respectively, which endowed them with great potential in controlled drug delivery. The intracellular DOX fluorescence intensity after the incubation with the laden micelles was different degrees weaker than that incubated with free DOX · HCl within 12 h, probably due to the slow DOX release from micelles. As the incubation time reached to 24 h, all the cells incubated with the laden micelles, especially SCM/DOX, demonstrated a stronger intracellular DOX fluorescence intensity than free DOX · HCl-cultured ones. More importantly, all the DOX-loaded micelles, especially SCM/DOX, exhibited potent antineoplastic efficacy in vitro, excellent serum albumin-tolerance stability, and satisfactory hemocompatibility. These encouraging data indicated that the loading micelles from nonlinear enantiomeric copolymers, especially SCM/DOX, might be promising in clinical systemic chemotherapy through intravenous injection.
Antineoplastic drug; Controlled delivery; Enantiomeric copolymers; Malignancy therapeutic; Stereocomplex micelle
The toxicity of anticancer agents and the difficulty in delivering drugs selectively to tumor cells pose a challenge in overcoming multidrug resistance (MDR). Recently, nanotechnology has emerged as a powerful tool in addressing some of the barriers to drug delivery, including MDR in cancer, by utilizing alternate routes of cellular entry and targeted delivery of drugs and genes. However, it is unclear whether doxorubicin (Dox) can be delivered by nanotechnologic approaches.
We asked whether (1) Dox-loaded lipid-functionalized dextran-based biocompatible nanoparticles (Dox/NP) can reverse MDR, (2) Dox/NP has more potent cytotoxic effect on MDR tumors than poly(ethylene glycol)-modified liposomal Dox (PLD), and (3) multidrug resistance protein 1 (MDR1) small interfering RNA loaded in these nanoparticles (siMDR1/NP) can modulate MDR.
To create stable Dox/NP and siMDR1/NP, we used two different lipid-modified dextran derivatives. The effect of Dox or Dox/NP was tested on drug-sensitive osteosarcoma (KHOS) and ovarian cancer (SKOV-3) cell cultures in triplicate and their respective MDR counterparts KHOSR2 and SKOV-3TR in triplicate. We determined the effects on drug retention, transfection efficacy of siMDR1/NP, and P-glycoprotein expression and the antiproliferative effect between Dox/NP and PLD in MDR tumor cells.
Fluorescence microscopy revealed efficient uptake of the Dox/NP and fluorescently tagged siMDR1/NP. Dox/NP showed five- to 10-fold higher antiproliferative activity at the 50% inhibitory concentration than free Dox in tumor cells. Dox/NP showed twofold higher activity than PLD in MDR tumor cells. siMDR1/NP (100 nM) suppressed P-glycoprotein expression in KHOSR2.
Dextran-lipid nanoparticles are a promising platform for delivering Dox and siRNAs.
Biocompatible dextran-based nanoparticles that are directly translatable to clinical medicine may lead to new potential therapeutics for reversing MDR in patients with cancer.
Supramolecular micelles as drug-delivery vehicles are generally unable to enter the nucleus of nondividing cells. In the work reported here, nuclear localization signal (NLS)-modified polymeric micelles were studied with the aim of improving nuclear drug delivery.
In this research, cholesterol-modified glycol chitosan (CHGC) was synthesized. NLS-conjugated CHGC (NCHGC) was synthesized and characterized using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX), an anticancer drug with an intracellular site of action in the nucleus, was chosen as a model drug. DOX-loaded micelles were prepared by an emulsion/solvent evaporation method. The cellular uptake of different DOX formulations was analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity of blank micelles, free DOX, and DOX-loaded micelles in vitro was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa and HepG2 cells.
The degree of substitution was 5.9 cholesterol and 3.8 NLS groups per 100 sugar residues of the NCHGC conjugate. The critical aggregation concentration of the NCHGC micelles in aqueous solution was 0.0209 mg/mL. The DOX-loaded NCHGC (DNCHGC) micelles were observed as being almost spherical in shape under transmission electron microscopy, and the size was determined as 248 nm by dynamic light scattering. The DOX-loading content of the DNCHGC micelles was 10.1%. The DOX-loaded micelles showed slow drug-release behavior within 72 hours in vitro. The DNCHGC micelles exhibited greater cellular uptake and higher amounts of DOX in the nuclei of HeLa cells than free DOX and DOX-loaded CHGC (DCHGC) micelles. The half maximal inhibitory concentration (IC50) values of free DOX, DCHGC, and DNCHGC micelles against HepG2 cells were 4.063, 0.591, and 0.171 μg/mL, respectively. Moreover, the IC50 values of free DOX (3.210 μg/mL) and the DCHGC micelles (1.413 μg/mL) against HeLa cells were nearly 6.96- and 3.07-fold (P < 0.01), respectively, higher than the IC50 value of the DNCHGC micelles (0.461 μg/mL).
The results of this study suggest that novel NCHGC micelles could be a potential carrier for nucleus-targeting delivery.
polymeric micelles; drug delivery; nucleus-targeting delivery
This study deals with the preparation and investigation of a nanoscale delivery system for the anticancer drug doxorubicin (DOX) using its complexation with polyanionic carbohydrate dextran sulfate (DS). Dynamic light scattering, SEM, and zeta potential determination were used to characterize nanocomplexes. DOX-DS complexation was studied in the presence of ethanol as a hydrogen-bond disrupting agent, NaCl as an electrostatic shielding agent, and chitosan as a positively charged polymer. Thermodynamics of DOX-DS interaction was studied using isothermal titration calorimetry (ITC). A dialysis method was applied to investigate the release profile of DOX from DOX-DS nanocomplexes. Spherical and smooth-surfaced DOX-DS nanocomplexes (250–500 nm) with negative zeta potential were formed at a DS/DOX (w/w) ratio of 0.4–0.6, with over 90% drug encapsulation efficiency. DOX when complexed with DS showed lower fluorescence emission and 480 nm absorbance plus a 15 nm bathometric shift in its visible absorbance spectrum. Electrostatic hydrogen bonding and π-π stacking interactions are the main contributing interactions in DOX-DS complexation. Thermal analysis of DOX-DS complexation by ITC revealed that each DOX molecule binds with 3 DS glycosyl monomers. Drug release profile of nanocomplexes showed a fast DOX release followed by a slow sustained release, leading to release of 32% of entrapped DOX within 15 days. DOX-DS nanocomplexes may serve as a drug delivery system with efficient drug encapsulation and also may be taken into consideration in designing DOX controlled-release systems.
chitosan; dextran; doxorubicin; nanocomplex; anticancer; drug delivery
A multifunctional unimolecular micelle made of a hyperbranched amphiphilic block copolymer was designed, synthesized, and characterized for cancer-targeted drug delivery and non-invasive positron emission tomography (PET) imaging in tumor-bearing mice. The hyperbranched amphiphilic block copolymer, Boltorn® H40-poly(L-glutamate-hydrazone-doxorubicin)-b-poly(ethylene glycol) (i.e., H40-P(LG-Hyd-DOX)-b-PEG), was conjugated with cyclo(Arg-Gly-Asp-D-Phe-Cys) peptides (cRGD, for integrin αvβ3 targeting) and macrocyclic chelators (1,4,7-triazacyclononane-N, N′, N″-triacetic acid [NOTA], for 64Cu-labeling and PET imaging) (i.e., H40-P(LG-Hyd-DOX)-b-PEG-OCH3/cRGD/NOTA, also referred to as H40-DOX-cRGD). The anti-cancer drug, doxorubicin (DOX) was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms (i.e., PLG) via a pH-labile hydrazone linkage to enable pH-controlled drug release. The unimolecular micelles exhibited a uniform size distribution and pH-sensitive drug release behavior. cRGD-conjugated unimolecular micelles (i.e., H40-DOX-cRGD) exhibited a much higher cellular uptake in U87MG human glioblastoma cells due to integrin αvβ3-mediated endocytosis than non-targeted unimolecular micelles (i.e., H40-DOX), thereby leading to a significantly higher cytotoxicity. In U87MG tumor-bearing mice, H40-DOX-cRGD-64Cu also exhibited a much higher level of tumor accumulation than H40-DOX-64Cu, measured by non-invasive PET imaging and confirmed by biodistribution studies and ex vivo fluorescence imaging. We believe that unimolecular micelles formed by hyperbranched amphiphilic block copolymers that synergistically integrate passive and active tumor-targeting abilities with pH-controlled drug release and PET imaging capabilities provide the basis for future cancer theranostics.
Unimolecular micelles; Drug delivery; Theranostic nanocarriers; Hyperbranched amphiphilic block; copolymer; Positron emission tomography (PET); Cyclic arginine-glycine-aspartic acid (cRGD); peptide
Star-shaped polymer micelles have good stability against dilution with water, showing promising application in drug delivery. In this work, biodegradable micelles made from star-shaped poly(å-caprolactone)/poly(ethylene glycol) (PCL/PEG) copolymer were prepared and used to deliver doxorubicin (Dox) in vitro and in vivo. First, an acrylated monomethoxy poly (ethylene glycol)-poly(å-caprolactone) (MPEG-PCL) diblock copolymer was synthesized, which then self-assembled into micelles, with a core-shell structure, in water. Then, the double bonds at the end of the PCL blocks were conjugated together by radical polymerization, forming star-shaped MPEG-PCL (SSMPEG-PCL) micelles. These SSMPEG-PCL micelles were monodispersed (polydispersity index = 0.11), with mean diameter of ≈25 nm, in water. Blank SSMPEG-PCL micelles had little cytotoxicity and did not induce obvious hemolysis in vitro. The critical micelle concentration of the SSMPEG-PCL micelles was five times lower than that of the MPEG-PCL micelles. Dox was directly loaded into SSMPEG-PCL micelles by a pH-induced self-assembly method. Dox loading did not significantly affect the particle size of SSMPEG-PCL micelles. Dox-loaded SSMPEG-PCL (Dox/SSMPEG-PCL) micelles slowly released Dox in vitro, and the Dox release at pH 5.5 was faster than that at pH 7.0. Also, encapsulation of Dox in SSMPEG-PCL micelles enhanced the anticancer activity of Dox in vitro. Furthermore, the therapeutic efficiency of Dox/SSMPEG-PCL on colon cancer mouse model was evaluated. Dox/SSMPEG-PCL caused a more significant inhibitory effect on tumor growth than did free Dox or controls (P < 0.05), which indicated that Dox/SSMPEG-PCL had enhanced anticolon cancer activity in vivo. Analysis with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) showed that Dox/SSMPEG-PCL induced more tumor cell apoptosis than free Dox or controls. These results suggested that SSMPEG-PCL micelles have promising application in doxorubicin delivery for the enhancement of anticancer effect.
drug delivery; star-shaped polymer; MPEG-PCL; CMC
Doxorubicin-loaded PEGylated liposomes (commercially available as DOXIL® or Lipodox®) were surface functionalized with a cell-penetrating peptide, octa-arginine (R8). For this purpose, R8-peptide was conjugated to the polyethylene glycol–dioleoyl phosphatidylethanolamine (PEG–DOPE) amphiphilic co-polymer. The resultant R8–PEG–PE conjugate was introduced into the lipid bilayer of liposomes at 2 mol% of total lipid amount via spontaneous micelle-transfer technique. The liposomal modification did not alter the particle size distribution, as measured by Particle Size Analyzer and transmission electron microscopy (TEM). However, surface-associated cationic peptide increased zeta potential of the modified liposomes. R8-functionalized liposomes (R8-Dox-L) markedly increased the intracellular and intratumoral delivery of doxorubicin as measured by flow cytometry and visualizing by confocal laser scanning microscopy (CLSM) compared to unmodified Doxorubicin-loaded PEGylated liposomes (Dox-L). R8-Dox-L delivered loaded Doxorubicin to the nucleus, being released from the endosomes at higher efficiency compared to unmodified liposomes, which had marked entrapment in the endosomes at tested time point of 1 h. The significantly higher accumulation of loaded drug to its site of action for R8-Dox-L resulted in improved cytotoxic activity in vitro (cell viability of 58.5 ± 7% for R8-Dox-L compared to 90.6 ± 2% for Dox-L at Dox dose of 50 μg/mL for 4 h followed by 24 h incubation) and enhanced suppression of tumor growth (348 ± 53 mm3 for R8-Dox-L, compared to 504 ± 54 mm3 for Dox-L treatment) in vivo compared to Dox-L. R8-modification has the potential for broadening the therapeutic window of pegylated liposomal doxorubicin treatment, which could lead to lower non-specific toxicity.
Doxorubicin; Liposomes; Octa-arginine; Drug delivery; Apoptosis
In this work, injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) were utilized to fabricate a bilayered osteochondral construct. Rabbit marrow mesenchymal stem cells (MSCs) were encapsulated with transforming growth factor-β3 (TGF-β3)-loaded MPs in the chondrogenic layer and cocultured with cells of different periods of osteogenic preculture (0, 3, 6 and 12 days) in the osteogenic layer to investigate the effects of TGF-β3 delivery and coculture on the proliferation and differentiation of cells in both layers. The results showed that, in the chondrogenic layer, TGF-β3 significantly stimulated chondrogenic differentiation of MSCs. Additionally, cells of various osteogenic preculture periods in the osteogenic layer, along with TGF-β3, enhanced gene expression for MSC chondrogenic markers to different extents. In the osteogenic layer, cells maintained their alkaline phosphatase activity during the coculture; however, mineralization was delayed by the presence of TGF-β3. Overall, this study demonstrated the fabrication of bilayered hydrogel composites that mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers, while illustrating that encapsulated cells of different degrees of osteogenic differentiation can significantly influence the chondrogenic differentiation of cocultured progenitor cells in both the presence and absence of chondrogenic growth factors.
bilayered hydrogel composites; mesenchymal stem cell; cell differentiation; coculture
To evaluate the cytotoxicity of poly(ethylene glycol)-block-poly(D,L-lactic acid) (PEG-PDLLA) nanovesicles loaded with doxorubicin (DOX) and the photosensitizer hematoporphyrin monomethyl ether (HMME) on human hepatocellular carcinoma HepG2 cells and to investigate potential apoptotic mechanisms.
PEG-PDLLA nanovesicles were simultaneously loaded with DOX and HMME (PEG-PDLLA-DOX-HMME), and PEG-PDLLA nanovesicles were loaded with DOX (PEG-PDLLA-DOX), HMME (PEG-PDLLA-HMME), or the PEG-PDLLA nanovesicle alone as controls. The cytotoxicity of PEG-PDLLA-DOX-HMME, PEG-PDLLA-DOX, PEG-PDLLA-HMME, and PEG-PDLLA against HepG2 cells was measured, and the cellular reactive oxygen species, percentage of cells with mitochondrial membrane potential depolarization, and apoptotic rate following treatment were determined.
Four nanovesicles (PEG-PDLLA-DOX-HMME, PEG-PDLLA-DOX, PEG-PDLLA-HMME, and PEG-PDLLA) were synthesized, and mean particle sizes were 175±18 nm, 154±3 nm, 196±2 nm, and 147±15 nm, respectively. PEG-PDLLA-DOX-HMME was more cytotoxic than PEG-PDLLA-DOX, PEG-PDLLA-HMME, and PEG-PDLLA. PEG-PDLLA-HMME-treated cells had the highest mean fluorescence intensity, followed by PEG-PDLLA-DOX-HMME-treated cells, whereas PEG-PDLLA-DOX- and PEG-PDLLA-treated cells had a similar fluorescence intensity. Mitochondrial membrane potential depolarization was observed in 54.2%, 59.4%, 13.8%, and 14.8% of the cells treated with PEG-PDLLA-DOX-HMME, PEG-PDLLA-HMME, PEG-PDLLA-DOX, and PEG-PDLLA, respectively. The apoptotic rate was significantly higher in PEG-PDLLA-DOX-HMME-treated cells compared with PEG-PDLLA-DOX- and PEG-PDLLA-HMME-treated cells.
The PEG-PDLLA nanovesicle, a drug delivery carrier, can be simultaneously loaded with two anticancer drugs (hydrophilic DOX and hydrophobic HMME). PEG-PDLLA-DOX-HMME cytotoxicity to HepG2 cells is significantly higher than the PEG-PDLLA nanovesicle loaded with DOX or HMME alone, and DOX and HMME have a synergistic effect against human hepatocellular carcinoma HepG2 cells.
polymeric vesicles; hematoporphyrin monomethyl ether; HMME; photodynamic therapy; PDT
This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF) (1–35 kDa)(a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally the product solution is filtered to remove byproduct salt, and the OPF product is twice crystallized, washed, and dried under vacuum. The reaction is affected by PEG molecular weight and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or UV-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, polymerize in situ, and undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.
oligo(poly(ethylene glycol) fumarate); OPF; polymer; tissue engineering; polymer synthesis; radical polymerization; hydrogel; PEG