Polyion Complex Nanoparticles Research Articles

Enzyme‐Responsive Polyion Complex Nanoparticles of Cationic Antimicrobials for Activatable Antibacterial Therapy

AbstractA self‐assembled “caging” strategy is presented for the safe delivery of potent cationic antimicrobials that suffer from non‐specific toxicity for the effective treatment of in vivo systemic bacterial infection. The key development here is a new block copolymer consisting of a poly(ethylene glycol) (PEG) stealth block and an anionic and lipase‐degradable block of poly(ɛ‐caprolactone) (PCL) and phosphonic acid‐bearing methacrylate copolymer, synthesized by hybrid copolymerization of methacrylate and cyclic esters. The anionic blocks electrostatically interact with cationic antimicrobials to form neutrally charged polyion complexes with the PEG blocks on the surface. The PCL component imparts the nanocomplex with biodegradability by bacterial secretory lipase. In the proof‐of‐concept study, cationic polyimidazolium that shows excellent antibacterial activity but severe toxicity is packaged by the block copolymer into nanocomplexes, which are stable in complex environments of high salt and protein concentrations and released the antimicrobials upon degradation of the copolymer by bacteria‐secreted lipase. The “caging” formulation of polyimidazolium eliminated its toxicity and led to highly effective bactericidal performance comparable to free polyimidazolium. This caging strategy does not require sophisticated chemical modification of cationic antimicrobials, offering a broadly applicable formulation strategy to overcome their common toxicity issue that has become a primary translational barrier.

Recent Progress in Polyion Complex Nanoparticles with Enhanced Stability for Drug Delivery.

Polyion complex (PIC) nanoparticles, including PIC micelles and PICsomes, are typically composed of poly(ethylene glycol) block copolymers coupled with oppositely charged polyelectrolytes or therapeutic agents via electrostatic interaction. Due to a simple and rapid preparation process with high drug-loading efficiency, PIC nanoparticles are beneficial to maintaining the chemical integrity and high biological activity of the loaded drugs. However, the stability of PIC nanoparticles can be disrupted in high-ionic-strength solutions because electrostatic interaction is the DRIVING force; these disruptions can thus impair drug delivery. Herein, we summarize the advances in the use of PIC nanoparticles for delivery of charged drugs, focusing on the different chemical and physical strategies employed to enhance their stability, including enhancing the charge density, crosslinking, increasing hydrophobic interactions, forming hydrogen bonds, and the development of PIC-based gels. In particular, we describe the use of PIC nanoparticles to load peptide antibiotics targeting antibiotic-resistant and biofilm-related diseases and the use of nanoparticles that load chemotherapeutics and gaseous donors for cancer treatment. Furthermore, the application of PIC nanoparticles as magnetic resonance imaging contrast agents is summarized for the first time. Therefore, this review is of great significance for advances in the use of polymeric nanoparticles for functional drug delivery.

Repurposing pentosan polysulfate sodium as hyaluronic acid linked polyion complex nanoparticles for the management of osteoarthritis: A potential approach

Osteoarthritis is still a disease burden for pharmaceutical scientists and strategy makers. It is associated with the chronic inflammation of joints especially weight-bearing joints like knee, hip, backbone, and phalanges. NSAIDs that are used for the management of inflammation associated with osteoarthritis have high side effects related to gastric upset, gastric ulcer, and long term treatment associated with liver and kidney damage. Nanotechnology has gained a huge scope for the management of arthritis as it can reach out to the deep inside the cell and alter cellular physiology as desired. The present study hypothesizes the use of polyion complex nanoparticles of hyaluronic acid linked Pentosan polysulfate sodium, a disease-modifying agent for the treatment of osteoarthritis administered through transdermal route. The hypothesis involves the use of drug repurposing as the drug was initially approved for interstitial cystitis, a condition of the urinary bladder associated with pain and swelling. Being very low oral bioavailability and gastric irritation profile, the transdermal route would be beneficial. To overcome the problem associated with the oral route, there is a need for the targeted approach that will particularly reach at inflammatory sites. Thereby transdermal delivery of hyaluronic acid linked Pentosan polysulfate sodium through polyion complex nanoparticle therapy will be a novel therapeutic approach to combat osteoarthritis.

Superoxide Dismutase 1 Nanoparticles (Nano-SOD1) as a Potential Drug for the Treatment of Inflammatory Eye Diseases.

Inflammatory eye diseases remain the most common clinical problem in ophthalmology. The secondary processes associated with inflammation, such as overproduction of reactive oxygen species (ROS) and exhaustion of the endogenous antioxidant system, frequently lead to tissue degeneration, vision blurring, and even blindness. Antioxidant enzymes, such as copper–zinc superoxide dismutase (SOD1), could serve as potent scavengers of ROS. However, their delivery into the eye compartments represents a major challenge due to the limited ocular penetration. This work presents a new therapeutic modality specifically formulated for the eye on the basis of multilayer polyion complex nanoparticles of SOD1 (Nano-SOD1), which is characterized by appropriate storage stability and pronounced therapeutic effect without side reactions such as eye irritation; acute, chronic, and reproductive toxicity; allergenicity; immunogenicity; mutagenicity even at high doses. The ability of Nano-SOD1 to reduce inflammatory processes in the eye was examined in vivo in rabbits with a model immunogenic uveitis—the inflammation of the inner vascular tract of the eye. It was shown during preclinical studies that topical instillations of Nano-SOD1 were much more effective compared to the free enzyme in decreasing uveitis manifestations. In particular, we noted statistically significant differences in such inflammatory signs in the eye as corneal and conjunctival edema, iris hyperemia, and fibrin clots. Moreover, Nano-SOD1 penetrates into interior eye structures more effectively than SOD itself and retains enzyme activity in the eye for a much longer period of time, decreasing inflammation and restoring antioxidant activity in the eye. Thus, the presented Nano-SOD1 can be considered as a potentially useful therapeutic agent for the treatment of ocular inflammatory disorders.

An organotypic model of high-grade serous ovarian cancer to test the anti-metastatic potential of ROR2 targeted Polyion complex nanoparticles.

High-grade serous ovarian cancer (HGSOC) is the most lethal gynaecological malignancy. Most patients are diagnosed at late stages when the tumour has metastasised throughout the peritoneal cavity. The Wnt receptor ROR2 has been identified as a promising therapeutic target in HGSOC, with limited targeting therapeutic options currently available. Small interfering RNA (siRNA)-based therapeutics hold great potential for inhibiting the function of specific biomarkers, however major challenges remain in efficient delivery and stability. The aim of this study was to investigate the ability of nanoparticles to deliver ROR2 siRNA into HGSOC cells, including platinum resistant models, and estimate the anti-metastatic effect via a 3D organotypic model for ovarian cancer. The nanoparticles were generated by conjugating poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA) of various chain length to bovine serum albumin (BSA), followed by the condensation of ROR2 siRNA into polyplexes, also termed polyion complex (PIC) nanoparticles. The toxicity and uptake of ROR2 siRNA PIC nanoparticles in two HGSOC cell lines, CaOV3 as well as its cisplatin resistant pair (CaOV3CisR), in addition to primary cells used for the 3D organotypic model were investigated. ROR2 knockdown at both transcriptional and translational levels were evaluated via real-time PCR and western blot analysis, respectively. Following 24 h incubation with the nanoparticles, functional assays were performed including proliferation (IncuCyte S3), transwell migration and 3D co-cultured transwell invasion assays. The PICs nanoparticles exhibited negligible toxicity in the paired CaOV3 cell lines or primary cells. Treating CaOV3 and CaOV3CisR cells with ROR2 siRNA containing PICs nanoparticles significantly inhibited migration and invasion ability. The biocompatible ROR2 siRNA conjugated PICs nanoparticles provide an innovative therapeutic option. ROR2 targeting therapy shows potential in treating HGSOC including platinum resistant forms.

Ternary Polypeptide Nanoparticles with Improved Encapsulation, Sustained Release, and Enhanced In Vitro Efficacy of Carfilzomib.

To develop a new nanoparticle formulation for a proteasome inhibitor Carfilzomib (CFZ) to improve its stability and efficacy for future in vivo applications. CFZ-loaded ternary polypeptide nanoparticles (CFZ/tPNPs) were prepared by using heptakis(6-amino-6-deoxy)-β-cyclodextrin(hepta-hydrochloride) (HaβCD) and azido-poly(ethylene glycol)-block-poly(L-glutamic acid sodium salt) (N3-PEG-PLE). The process involved ternary (hydrophobic/ionic/supramolecular) interactions in three steps: 1) CFZ was entrapped in the cavity of HaβCD by hydrophobic interaction, 2) the drug-cyclodextrin inclusion complexes were mixed with N3-PEG-PLE to form polyion complex nanoparticles, and 3) the nanoparticles were modified with fluorescent dyes (AFDye 647) for imaging and/or epithelial cell adhesion molecule (EpCAM) antibodies for cancer cell targeting. CFZ/tPNPs were characterized for particle size, surface charge, drug release, stability, intracellular uptake, proteasome inhibition, and in vitro cytotoxicity. tPNPs maintained an average particle size of 50nm after CFZ entrapment, EpCAM conjugation, and freeze drying. tPNPs achieved high aqueous solubility of CFZ (>1mg/mL), sustained drug release (t1/2 = 6.46h), and EpCAM-mediated cell targeting, which resulted in increased intracellular drug accumulation, prolonged proteasome inhibition, and enhanced cytotoxicity of CFZ in drug-resistant DLD-1 colorectal cancer cells. tPNPs improved stability and efficacy of CFZ in vitro, and these results potentiate effective cancer treatment using CFZ/tPNPs in future vivo studies.

Fast-Bactericidal Effect of Polyion Complex Nanoparticles on Gram-Negative Bacteria

There is urgent need for effective bactericidal agents for use in real commercial formulations since many old disinfectants, such as halogenated compounds, are now banned. Cationic polymers may have good bactericidal properties in pure water or buffer, but typically become ineffective in the presence of anionic surfactants that are widely used in many commercial formulations. Here, we discover that polyion complex (PIC) nanoparticles formed by cationic polymers of Poly(3-AcrylaMidoPropyl) TriMethylAmmonium chloride (PAMPTMA) in the presence of anionic surfactants display promising fast-bactericidal effect (> 99.99 % killing within 10-min treatment) on Gram-negative Escherichia coli (E. coli, ATCC® 8739™). To examine the influence of hydrophobicity on bactericidal property, we synthesize PAMPTMA-b-Poly(Butyl MethAcrylate) and discover that increasing hydrophobicity has little influence on bactericidal property of PIC nanoparticles. Mechanism study shows that cationic PIC nanoparticles rapidly cause signifi...

Encapsulation of tissue plasminogen activator in pH-sensitive self-assembled antioxidant nanoparticles for ischemic stroke treatment – Synergistic effect of thrombolysis and antioxidant –

The medical treatment for stroke has advanced greatly in recent years. Thrombolytic therapy with tissue plasminogen activator (t-PA) is one of the mainstream treatments, but it still has many problems, including short half-life, and t-PA-induced reperfusion and oxidative injuries. To broaden the therapeutic window of t-PA and reduce its associated oxidative stress after reperfusion, t-PA-installed, nitroxide radical-containing, self-assembled polyion complex nanoparticles (t-PA@iRNP) were designed. Encapsulation of t-PA in the self-assembled antioxidant nanoparticles improved its bioavailability and extended its therapeutic window. To suppress reactive oxygen species (ROS) in the ischemic penumbra area, the low-molecular-weight nitroxide antioxidant 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl was covalently conjugated with the nanoparticle matrix, thus suppressing oxidative damage in the brain after reperfusion. t-PA and nitroxide radicals were confined and protected in the core of t-PA@iRNP, thereby preventing their rapid metabolism and excretion out of the body after systemic circulation for prolonged period. The nano-sized formulation prevented non-specific internalization of t-PA@iRNP in healthy cells, thereby preserving the normal function of redox reactions in the cells, especially important redox reactions such as electron transport chains. This improved pharmacological performance of t-PA@iRNP remarkably extended the in vivo half-life of t-PA in systemic circulation. Using a mouse model of photo-thrombotic middle cerebral artery occlusion, we found that t-PA@iRNP treatment, compared with naked t-PA, void iRNP, or t-PA@niRNP (non-ROS scavenging nanoparticle as a control), significantly suppressed increases in cerebral infarct volume and improved neurological deficit after brain ischemia. t-PA-induced subarachnoid hemorrhage was also suppressed by t-PA@iRNP treatment through elimination of overproduced ROS. Based on these data, t-PA@iRNP presents therapeutic potential through synergistic effect of thrombolysis and antioxidant effects for preventing and treating ischemia-reperfusion injury.

Structural Determinants of the Stability of Enzyme-Responsive Polyion Complex Nanoparticles Targeting Pseudomonas aeruginosa's Elastase.

Here, we report how the stability of polyion complex (PIC) particles containing Pseudomonas aeruginosa’s elastase (LasB) degradable peptides and antimicrobial poly(ethylene imine) is significantly improved by careful design of the peptide component. Three LasB‐degradable peptides are reported herein, all of them carrying the LasB‐degradable sequence −GLA− and for which the number of anionic amino acids and cysteine units per peptide were systematically varied. Our results suggest that while net charge and potential to cross‐link via disulfide bond formation do not have a predictable effect on the ability of LasB to degrade these peptides, a significant effect of these two parameters on particle preparation and stability is observed. A range of techniques has been used to characterize these new materials and demonstrates that increasing the charge and cross‐linking potential of the peptides results in PIC particles with better stability in physiological conditions and upon storage. These results highlight the importance of molecular design for the preparation of PIC particles and should underpin the future development of these materials for responsive drug delivery.

Polymyxin B containing polyion complex (PIC) nanoparticles: Improving the antimicrobial activity by tailoring the degree of polymerisation of the inert component

Here, we describe the preparation and characterisation of polyion complex (PIC) nanoparticles containing last resort antimicrobial polymyxin B (Pol-B). PIC nanoparticles were prepared with poly(styrene sulphonate) (PSS) as an inert component, across a range of degrees of polymerisation to evaluate the effect that multivalency of this electrolyte has on the stability and antimicrobial activity of these nanoparticles. Our results demonstrate that while nanoparticles prepared with longer polyelectrolytes are more stable under simulated physiological conditions, those prepared with shorter polyelectrolytes have a higher antimicrobial activity. Tailoring the degree of polymerisation and the ratio of the components we have been able to identify a formulation that shows a sustained inhibitory effect on the growth of P. aeruginosa and can reduce the number of viable colonies of this pathogen over 10,000 times more effectively than our previously reported formulation.

Polyion complex (PIC) particles: Preparation and biomedical applications

Oppositely charged polyions can self-assemble in solution to form colloidal polyion complex (PIC) particles. Such nanomaterials can be loaded with charged therapeutics such as DNA, drugs or probes for application as novel nanomedicines and chemical sensors to detect disease markers. A comprehensive discussion of the factors affecting PIC particle self-assembly and their response to physical and chemical stimuli in solution is described herein. Finally, a collection of key examples of polyionic nanoparticles for biomedical applications is discussed to illustrate their behaviour and demonstrate the potential of PIC nanoparticles in medicine.

Induction of cell self-organization on weakly positively charged surfaces prepared by the deposition of polyion complex nanoparticles of thermoresponsive, zwitterionic copolymers.

We have developed inducible cell self-organization through weakly positively charged culture surfaces. In this study, a thermoresponsive and zwitterionic copolymer comprised of N,N-dimethylaminoethyl methacrylate (DMAEMA) and methacrylic acid (MA) (PDMAEMA-co-PMA; Mn: ∼9.7 × 104 g/mol; PDMAEMA/PMA ratio: 10) was designed for inducing cell self-organization. The copolymer formed single polymer-derived polyion complex (sPIC) nanoparticles following dissolution in an aqueous solution. The sPIC nanoparticles had a positive charge (ca. 25 mV). Self-organization occurred in adipose-derived vascular stromal cell monolayers cultivated on sPIC-deposited surfaces. There were dramatic morphological changes of these cells with the formation of capillary-like networks and single-cell aggregates with little cytotoxicity. This was a significant improvement compared with cells grown on previously developed surfaces deposited with PIC, a mixture of PDMAEMA and plasmid DNA. Thus, sPICs of PDMAEMA-co-PMA may allow for the accurate evaluation of a variety of cell behaviors with less cytotoxicity, and may facilitate additional potential medical applications such as cell-based therapy and drug discovery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1009-1015, 2017.

Enzyme-responsive polyion complex (PIC) nanoparticles for the targeted delivery of antimicrobial polymers.

Here we present new enzyme-responsive polyion complex (PIC) nanoparticles prepared from antimicrobial poly(ethylene imine) and an anionic enzyme-responsive peptide targeting Pseudomonas aeruginosa's elastase. The synthetic conditions used to prepare these nanomaterials allowed us to optimise particle size and charge, and their stability under physiological conditions. We demonstrate that these enzyme responsive PIC nanoparticles are selectively degraded in the presence of P. aeruginosa elastase without being affected by other endogenous elastases. This enzyme-responsive PIC particle can exert an elastase-specific antimicrobial effect against P. aeruginosa without affecting non-pathogenic strains of these bacteria. These targeted enzyme-responsive PIC nanoparticles constitute a novel platform for the delivery of antimicrobial peptides and polymers, and can be a powerful tool in the current race against antimicrobial resistance.

Induction of potent adaptive immunity by the novel polyion complex nanoparticles.

The development of effective and simple methods of vaccine preparation is desired for the prophylaxis and treatment of a variety of infectious diseases and cancers. We have created novel polyion complex (PIC) nanoparticles (NPs) composed of amphiphilic anionic biodegradable poly(γ-glutamic acid) (γ-PGA) and cationic polymers as a vaccine adjuvant. PIC NPs can be prepared by mixing γ-PGA-graft-l-phenylalanine ethylester (γ-PGA-Phe) polymer with cationic polymer in phosphate-buffered saline. We examined the efficacy of PIC NPs for antigen delivery and immunostimulatory activity in vitro and in vivo. PIC NPs enhanced the uptake of ovalbumin (OVA) by dendritic cells (DCs) and subsequently induced DC maturation. The immunization of mice with OVA-carrying PIC NPs induced potent and antigen-specific cellular and humoral immunity. Since PIC NPs can be created with water-soluble anionic γ-PGA-Phe and a cationic polymer by simple mixing in the absence of any organic solvents, PIC NPs may have potential as a novel candidate for an effective antigen carrier and vaccine adjuvant.

HTLV-1 Tax peptide-carrying polyion complex nanoparticles induce potent cellular immunity in mice

Development of safe and effective vaccines is important for controlling a variety of infectious diseases, including retroviral infections. The induction of cytotoxic T lymphocytes (CTLs) is a promising strategy for elimination of infected cells. Polyion complex (PIC) nanoparticles have been created using anionic biodegradable poly(γ-glutamic acid) (γ-PGA) and cationic protamine. Amphiphilic graft copolymers, consisting of γ-PGA and l-phenylalanine (l-Phe) hydrophobic side chain, were synthesized by grafting l-Phe to γ-PGA. The PIC nanoparticles were prepared by mixing the graft copolymers with protamine in phosphate buffered saline. In this study, antigen peptide-carrying PIC nanoparticles were examined for their effect on the induction of antigen-specific cellular immunity in mice. The antigen-specific CTL response was evaluated by intracellular cytokine staining and IFN-γ ELISPOT assay. The immunization with PIC nanoparticles carrying HTLV-1 Tax peptide could induce the expansion of Tax-specific CD8+ T cells. In contrast, no such induction of the antigen-specific CD8+ T cells was observed, when mice were immunized with the peptide alone or peptide plus an aluminum adjuvant. These results suggest that the Tax peptide-carrying PIC nanoparticles are capable of inducing cellular immune responses and may have potential as an effective vaccine adjuvant for anti-HTLV-1 vaccines.

Rational Design of Polyion Complex Nanoparticles to Overcome Cisplatin Resistance in Cancer Therapy

Rationally designed PIC nanoparticles as next-generation delivery system: we have developed a core-shell-corona PIC nanoparticle (⊕) NP/Pt@PPC-DA as a next-generation delivery system. (⊕) NP/Pt@PPC-DA exhibits prolonged circulation and enhanced drug accumulation in tumors. Subsequently, tumor pH leads to the release of (⊕) NP/Pt, which facilitates cellular uptake followed by rapid intracellular cisplatin release. Using this delivery strategy cisplatin-resistant tumor growth in a murine xenograft model has been successfully suppressed.

The effect of electrically charged polyion complex nanoparticle-coated surfaces on adipose-derived stromal progenitor cell behaviour

Surface characteristics of biomaterials such as wettability, rigidity, roughness, and electrical charge affect the fate of transplanted cells such as progenitor cells or stem cells for use in regenerative medicine. Of these, the effects of surface electrical charges on cellular behaviour such as adhesion, proliferation, and differentiation are not well understood. We prepared precisely charged culture surfaces ranging from −28 mV to +21 mV, simply by surface deposition of polyion complex nanoparticles prepared by mixing a positively charged thermoresponsive homopolymer, poly(N,N-dimethylaminoethyl methacrylate), with negatively charged plasmid DNA at various charge ratios. Drastic morphological changes of adipose-derived vascular progenitor cells were generated on the positively charged surface of organized forms at +19 mV. Capillary-like networks or single aggregates of these cells were selectively created depending on cell seeding density. Our findings offer new insights that may aid develop stem cell-processing techniques for use in regenerative medicine.

A new kind of polyion complex nanoparticles and the covalent drug-loading pattern for doxorubicin and pH-controlled release

The one-pot synthesis method was developed for the preparation of complex nanoparticles with a narrow size distribution and stable morphology. The vinyl monomers of (2-dimethylamino)ethyl methacrylate (DEMA) and diacetone acrylamide (DAA) were copolymerized in the presence of alginic acid in an aqueous solution without any organic solvents or surfactants, yielding stable complex nanoparticles in one-pot synthesis. The nanoparticle was composed of the complex of poly(DEMA-co-DAA) and alginic acid. The complex was formed via electrostatic interaction between polycations of DEMA and polyanions of alginate. The residual alginate segment around the core formed the shell of the nanoparticles. The average diameter of the nanoparticles varied from 120 to 213 nm when the molar percentage of DAA changed from 0.5 to 0 with respect to DEMA. The anti-cancer drug doxorubicin could be loaded onto the nanoparticles with a high-loading efficiency through the formation of polymer–drug conjugate. The drug release could be controlled by adjusting the pH value of the medium.

Self-assembly of polyion complex nanocarriers from ammonium glycyrrhizinate and monomethoxy poly(ethylene glycol)-b-poly(γ-amino-ε-caprolactone).

A novel polyion complex (PIC) drug carrier was developed via electrostatic self-assembly in aqueous media based on our previous copolymer methoxy poly(ethylene glycol)-b-poly(γ-amino-ε-caprolactone) (mPEG-b-PACL) and anti-hepatotoxic drug ammonium glycyrrhizinate (AMG). The formation and the physicochemical properties of the polyion complex nanoparticles in aqueous solution were investigated by various methods. The results demonstrated that AMG was encapsulated successfully and the nanoparticles had a core-shell or tri-layer spherical structure based on the length of the PACL. The AMG/PACL PIC also displayed a high loading capacity and encapsulation efficiency of up to 41.8% and 91.7%, respectively. Furthermore, the nanoparticles enabled a continuous and sustained in vitro AMG release and the release half time was about 3 times that of the free AMG's. Results obtained by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and fluorescence microscopy revealed that AMG loaded nanoparticles demonstrated greater prevention of liver cell apoptosis induced by lipopolysaccharide (LPS). These findings indicated that methoxy poly(ethylene glycol) (mPEG)-b-poly(γ-amino-ε-caprolactone) could be an efficient AMG carrier and could even be used as a delivery template for some other negatively charged water-soluble drugs or gene delivery systems.

Dual growth factor-releasing nanoparticle/hydrogel system for cartilage tissue engineering

In order to induce the chondrogenesis of mesenchymal stem cells (MSCs) in tissue engineering, a variety of growth factors have been adapted and encouraging results have been demonstrated. In this study, we developed a delivery system for dual growth factors using a gelation rate controllable alginate solution (containing BMP-7) and polyion complex nanoparticles (containing TGF-beta(2)) to be applied for the chondrogenesis of MSCs. The dual growth factors (BMP-7/TGF-beta(2))-loaded nanoparticle/hydrogel system showed a controlled release of both growth factors: a faster release of BMP-7 and a slower release of TGF-beta(2), ca., approximately 80 and 30% release at the end of an incubation period (21 days), respectively, which may be highly desirable for chondrogenic differentiation of MSCs. On the contrary, the release of each growth factor from the dual growth factors-loaded hydrogel (without the nanoparticles) was much slower than that of the nanoparticle/hydrogel system, approximately 36% (BMP-7) and 16% (TGF-beta(2)) for 21 days, and this is more than likely attributed to the aggregation between growth factors during the hydrogel fabrication step. The nanoparticle/hydrogel system with separate growth factor loading may provide desirable growth factor delivery kinetics for cartilage regeneration, as well as the chondrogenesis of MSCs.