Tumor-specific cytolysis by peptide-conjugated echogenic polymer micelles.

Interest in multifunctional polymer nanoparticles for targeted delivery of anti-cancer drugs has grown significantly in recent years. In this study, tumor-targeting echogenic polymer micelles were prepared from poly(ethylene glycol) methyl ether-alkyl carbonate (mPEG-AC) derivatives, and their potential in cancer therapy was assessed. Various mPEG derivatives with carbonate linkages were synthesized via an alkyl halide reaction between mPEG and alkyl chloroformate. Micelle formation using polymer amphiphiles in aqueous media and the subsequent carbon dioxide (CO2) gas generation from the micelles was confirmed. Their ability to target neuroblastoma was substantially enhanced by incorporating the rabies virus glycoprotein (RVG) peptide. RVG-modified gas-generating micelles significantly inhibited tumor growth in a tumor-bearing mouse model owing to CO2 gas generation within tumor cells and resultant cytolytic effects, showing minimal side effects. The development of multifunctional polymer micelles may offer a promising therapeutic approach for various diseases, including cancer.

Multi-Functional Polymer Nanoparticles with Enhanced Adipocyte Uptake and Adipocytolytic Efficacy.

Multi-functional polymer nanoparticles have been widely utilized to improve cellular uptake and enhance therapeutic efficacy. In this study, it is hypothesized that the cellular uptake of poly(D,L-lactide-co-glycolide) (PLG) nanoparticles loaded with calcium carbonate minerals into adipocytes can be improved by covalent modification with nona-arginine (R9 ) peptide. It is further hypothesized that the internalization mechanism of R9 -modified PLG nanoparticles by adipocytes may be contingent on the concentration of R9 peptide present in the nanoparticles. R9 -modified PLG nanoparticles followed the direct penetration mechanism when the concentration of R9 peptide in the nanoparticles reached 38 µM. Notably, macropinocytosis is the major endocytic mechanism when the R9 peptide concentration is ≤ 26 µM. The endocytic uptake of the nanoparticles effectively generated carbon dioxide gas at an endosomal pH, resulting in significant adipocytolytic effects in vitro, which are further supported by the findings in an obese mouse model induced by high-fat diet. Gas-generating PLG nanoparticles, modified with R9 peptide, demonstrated localized reduction of adipose tissue (reduction of 13.1%) after subcutaneous injection without significant side effects. These findings highlight the potential of multi-functional polymer nanoparticles for the development of effective and targeted fat reduction techniques, addressing both health and cosmetic considerations.

Adipocytolytic Polymer Nanoparticles for Localized Fat Reduction.

The demand for body fat reduction is increasing. However, conventional lipolytic approaches fail to control adipose tissue reduction and cause severe side effects in adjacent nonadipose tissues. A strategy to specifically reduce subcutaneous fat using adipocytolytic polymer nanoparticles in a minimally invasive manner is reported here. The polymer nanoparticles are designed to generate carbon dioxide gas when selectively absorbed by adipocytes. The carbon dioxide gas generated within late endosomes/lysosomes induces adipocytolysis, thereby reducing the number of cells. Localized injection of the adipocytolytic nanoparticles substantially reduces subcutaneous fat in a high-fat diet-induced obese mouse model, without significant changes in hematological or serum biochemical parameters. The adipocytolytic efficacy of the nanoparticles is also evaluated in a porcine model. This strategy addresses the need to develop safe and effective adipocytolytic agents using functional polymer nanoparticles.

Intracellular Glucose-Depriving Polymer Micelles for Antiglycolytic Cancer Treatment.

A new anticancer strategy to exploit abnormal metabolism of cancer cells rather than to merely control the drug release or rearrange the tumor microenvironment is reported. An antiglycolytic amphiphilic polymer, designed considering the unique metabolism of cancer cells (Warburg effect) and aimed at the regulation of glucose metabolism, is synthesized through chemical conjugation between glycol chitosan (GC) and phenylboronic acid (PBA). GC-PBA derivatives form stable micellar structures under physiological conditions and respond to changes in glucose concentration. Once the micelles accumulate at the tumor site, intracellular glucose capture occurs, and the resultant energy deprivation through the inhibition of aerobic glycolysis remarkably suppresses tumor growth without significant side effects in vivo. This strategy highlights the need to develop safe and effective cancer treatment without the use of conventional anticancer drugs.

Three-dimensional printing of hyaluronate-based self-healing ferrogel with enhanced stretchability.

Hydrogels have been frequently employed for three-dimensional (3D) printing, which is a promising tool for fabricating sophisticated structures useful in many biomedical applications. Ferrogels prepared by combining magnetic nanoparticles with hydrogels also have potential in biomedical engineering because of the responsiveness to a magnetic field and remotely controllable properties. However, typical ferrogels, especially those prepared from natural polysaccharides, have limitations concerning their mechanical properties and the fabrication method of complex structures owing to their rigid and brittle properties. In this study, 3D printable and stretchable ferrogel was designed and prepared to overcome these limitations. Hyaluronic acid (HA) derivatives such as hydrazide-modified HA (hHA) and oxidized HA (oHA) were used as the base materials for gel preparation. Self-healing oHA/hHA hydrogels were prepared by the addition of adipic acid dihydrazide (ADH). Self-healing ferrogels with 3D printability were prepared by adding superparamagnetic iron oxide nanoparticles (SPIONs) to oHA/hHA/ADH hydrogels, which improved the stretchability owing to the double network formation (2.1 times its original length). Various 3D constructs were fabricated by an extrusion-based printing method using ferrogel (structural integrity = 94.3 ± 1.5%). The potential to fabricate hydrogel/ferrogel hybrid constructs for tissue engineering was also investigated. This approach for developing customized 3D constructs using magnetic field-responsive and 3D printable hydrogel systems may find useful applications in tissue engineering approaches.

Rapid Assessment of Di(2-ethylhexyl) Phthalate Migration from Consumer PVC Products.

Poly(vinyl chloride) (PVC) is widely used to produce various consumer goods, including food packaging, toys for children, building materials, and cosmetic products. However, despite their widespread use, phthalate plasticizers have been identified as endocrine disruptors, which cause adverse health effects, thus leading to increasing concerns regarding their migration from PVC products to the environment. This study proposed a method for rapidly measuring the migration of phthalates, particularly di(2-ethylhexyl) phthalate (DEHP), from PVC products to commonly encountered liquids. The release of DEHP under various conditions, including exposure to aqueous and organic solvents, different temperatures, and household microwaves, was investigated. The amount of DEHP released from both laboratory-produced PVC films and commercially available PVC products was measured to elucidate the potential risks associated with its real-world applications. Furthermore, tests were performed to evaluate cytotoxicity using estrogen-dependent and -independent cancer cell lines. The results revealed a dose-dependent impact on estrogen-dependent cells, thus emphasizing the potential health implications of phthalate release. This comprehensive study provides valuable insights into the migration patterns of DEHP from PVC products and forms a basis for further research on the safety of PVC and plasticizers.

Stretchable and self-healable hyaluronate-based hydrogels for three-dimensional bioprinting.

Hydrogels have been widely exploited as inks for three-dimensional (3D) bioprinting, a useful technique for building complex biological structures with living cells. However, hydrogels have inherently limited mechanical properties (e.g., brittleness) and printability. Thus, we hypothesized that hyaluronate-based hydrogels with stretchable and self-healing properties would be useful for 3D bioprinting. Oxidized hyaluronate (oHA) and hydrazide-modified hyaluronate (hHA) formed stretchable and flexible hydrogels because of double network formation via chemical cross-linking (i.e., acylhydrazone bond formation) and physical cross-linking (i.e., charge interaction). The addition of adipic acid dihydrazide (ADH) to oHA/hHA hydrogels enhanced the self-healing capability of the gels, which were useful for fabricating 3D constructs with various shapes maintaining their stretchability even after 3D printing (about two times its original length). ATDC5 cells were viable within the 3D-printed constructs in vitro. This hydrogel system, consisting of hyaluronic acid (HA)-based polymers, may have potential for many tissue engineering applications via 3D bioprinting.

In vitro culture of hematopoietic stem cell niche using angiopoietin-1-coupled alginate hydrogel.

Stem cells exist and maintain their quiescence and pluripotency in stem cell niche. Here, we hypothesized that regulation of cell-cell interactions using a polymeric scaffold as synthetic extracellular matrix (ECM) could be critical in creating a hematopoietic stem cell (HSC) niche in vitro. Angiopoietin-1 (Ang1) binds to the tyrosine kinase receptor (Tie2), and regulation of the Tie2/Ang1 interaction is important in maintaining the quiescence of HSCs in vivo. Alginate hydrogel was thus modified with Ang1 as a synthetic ECM to mimic the HSC niche. Long-term HSCs (CD34-, CD135-, and CD150+) were isolated from mouse femurs and cultured on Ang1-modified alginate hydrogel. The percentage of LT-HSCs in G0 phase was 46.8 ± 1.8%, which was comparable to that of LT-HSCs co-cultured with osteoblasts (46.8 ± 2.1%). Ang1-coupled alginate gels were useful to provide a niche for HSC quiescence without a co-culture system. Polymeric scaffolds containing biomimetic and cell-instructive characteristics for stem cell phenotype regulation might help create HSC niches in vitro and be useful to engineer tissues and transplant stem cells.

Three-dimensional bioprinting of polysaccharide-based self-healing hydrogels with dual cross-linking.

Three-dimensional (3D) bioprinting technique is useful to fabricate constructs with functional and biological structures for various biomedical applications. Oxidized hyaluronate (OHA) and glycol chitosan (GC) can form autonomous self-healing hydrogels when adipic acid dihydrazide (ADH) is used. We demonstrate that hyaluronate-alginate hybrid (HAH) polymers can be used for secondary physical cross-linking of OHA/GC/ADH hydrogel with calcium ions after 3D printing. The molecular weight of hyaluronate can be varied while keeping the molecular weight of alginate in HAH. The mechanical stiffness and stability of gels after 3D printing are strongly dependent on the molecular weight of HAH at the same cross-linking density. In vitro chondrogenic differentiation of ATDC5 cells encapsulated in 3D-printed constructs is dependent on the molecular weight of HAH in gels. This dual cross-linking system consisting of naturally occurring biocompatible polysaccharides may have potential in the 3D bioprinting of custom-made scaffolds for tissue engineering applications.

In Vitro Cellular Uptake and Transfection of Oligoarginine-Conjugated Glycol Chitosan/siRNA Nanoparticles.

Chitosan and its derivatives have been extensively utilized in gene delivery applications because of their low toxicity and positively charged characteristics. However, their low solubility under physiological conditions often limits their application. Glycol chitosan (GC) is a derivative of chitosan that exhibits excellent solubility in physiological buffer solutions. However, it lacks the positive characteristics of a gene carrier. Thus, we hypothesized that the introduction of oligoarginine peptide to GC could improve the formation of complexes with siRNA, resulting in enhanced uptake by cells and increased transfection efficiency in vitro. A peptide with nine arginine residues and 10 glycine units (R9G10) was successfully conjugated to GC, which was confirmed by infrared spectroscopy, 1H NMR spectroscopy, and elemental analysis. The physicochemical characteristics of R9G10-GC/siRNA complexes were also investigated. The size and surface charge of the R9G10-GC/siRNA nanoparticles depended on the amount of R9G10 coupled to the GC. In addition, the R9G10-GC/siRNA nanoparticles showed improved uptake in HeLa cells and enhanced in vitro transfection efficiency while maintaining low cytotoxicity determined by the MTT assay. Oligoarginine-modified glycol chitosan may be useful as a potential gene carrier in many therapeutic applications.

3D Printing of dynamic tissue scaffold by combining self-healing hydrogel and self-healing ferrogel.

Hydrogels have been widely utilized in tissue engineering applications as functional and biological synthetic extracellular matrices (ECMs) can be created with gels. However, typical hydrogels cannot be exploited in 3D printing, especially in extrusion printing, unless post-cross-linking after printing is provided. Additionally, dynamic tissue scaffolds that can mimic ECM environments in the body have been demonstrated to be useful in tissue engineering. Here, we hypothesized that a 3D-printed dynamic tissue scaffold could be fabricated by combining self-healing hydrogel and self-healing ferrogel without post-cross-linking, which could be useful for the regulation of cell phenotype under magnetic stimulation. Hydrogels were formed from oxidized sodium hyaluronate and glycol chitosan, and adipic acid dihydrazide was additionally utilized for self-healing behavior of the gel. Superparamagnetic iron oxide nanoparticles (SPIONs) were also used to prepare a magnetically responsive hydrogel system (i.e., ferrogel). Physicochemical properties, cytotoxicity, and printability of the self-healing hydrogel/ferrogel system fabricated by a 3D printing process, were investigated. Dimensional changes in a tissue scaffold were achieved by the application of a magnetic field. Interestingly, chondrogenic differentiation of ATDC5 cells cultured within the dynamic tissue scaffold was enhanced by applying a magnetic field in vitro. This approach may be useful for fabricating dynamic tissue scaffolds by a 3D printing method for tissue engineering applications.

3D Printing of Polysaccharide-Based Self-Healing Hydrogel Reinforced with Alginate for Secondary Cross-Linking.

Three-dimensional (3D) bioprinting has been attractive for tissue and organ regeneration with the possibility of constructing biologically functional structures useful in many biomedical applications. Autonomous healing of hydrogels composed of oxidized hyaluronate (OHA), glycol chitosan (GC), and adipic acid dihydrazide (ADH) was achieved after damage. Interestingly, the addition of alginate (ALG) to the OHA/GC/ADH self-healing hydrogels was useful for the dual cross-linking system, which enhanced the structural stability of the gels without the loss of their self-healing capability. Various characteristics of OHA/GC/ADH/ALG hydrogels, including viscoelastic properties, cytotoxicity, and 3D printability, were investigated. Additionally, potential applications of 3D bioprinting of OHA/GC/ADH/ALG hydrogels for cartilage regeneration were investigated in vitro. This hydrogel system may have potential for bioprinting of a custom-made scaffold in various tissue engineering applications.

On/off switchable physical stimuli regulate the future direction of adherent cellular fate.

The utilization of cell-manipulating techniques reveals information about biological behaviors suited to address a wide range of questions in the field of life sciences. Here, we introduced an on/off switchable physical stimuli technique that offers precise stimuli for reversible cell patterning to allow regulation of the future direction of adherent cellular behavior by leveraging enzymatically degradable alginate hydrogels with defined chemistry and topography. As a proof of concept, targeted muscle cells adherent to TCP exhibited a reshaped structure when the hydrogel-based physical stimuli were applied. This simple tool offers easy manipulation of adherent cells to reshape their morphology and to influence future direction depending on the characteristics of the hydrogel without limitations of time and space. The findings from this study are broadly applicable to investigations into the relationships between cells and physiological extracellular matrix environments as well as has potential to open new horizons for regenerative medicine with manipulated cells.

Regulation of the Viscoelastic Properties of Hyaluronate-Alginate Hybrid Hydrogel as an Injectable for Chondrocyte Delivery.

Modulation of the viscoelastic properties of hydrogels is critical in tissue engineering applications. In the present study, a hyaluronate-alginate hybrid (HAH) was synthesized by introducing alginate to the hyaluronate backbone with varying molecular weights (700-2500 kDa), and HAH hydrogels were prepared in the presence of calcium ions at the same cross-linking density. The storage shear moduli of the HAH hydrogels increased with the concomitant increase in the molecular weight of hyaluronate in the HAH polymer. The HAH hydrogels were also modified with arginine-glycine-aspartic acid (RGD) and histidine-alanine-valine (HAV) peptides to enhance cell-matrix and cell-cell interactions, respectively. The chondrogenic differentiation of ATDC5 cells encapsulated within the HAH hydrogels was enhanced with the increase in the storage shear moduli of the gels in vitro as well as in vivo. This approach of regulating the viscoelastic properties of hydrogels using polymers of varying molecular weights at the same cross-linking density may prove to be useful in various tissue engineering applications including cartilage regeneration.

3D printing of self-healing ferrogel prepared from glycol chitosan, oxidized hyaluronate, and iron oxide nanoparticles.

Hydrogel systems that show self-healing ability after mechanical damage are receiving increasing attention. However, self-healing hydrogels suitable for biomedical applications are limited owing to complex preparation methods. Furthermore, few studies have demonstrated the self-healing property of ferrogels. In this study, we demonstrated that glycol chitosan (GC) and oxidized hyaluronate (OHA) can be used to form a self-healing ferrogel in the presence of superparamagnetic iron oxide nanoparticles (SPIONs) without additional chemical cross-linkers. The overall characteristics of GC/OHA/SPION ferrogel varied based on the GC/OHA ratio, SPION content, and total polymer concentration. Interestingly, GC/OHA/SPION ferrogel was used to fabricate 3D-printed constructs of various shapes via an extrusion printing method. These constructs were responsive to the magnetic field, suggesting their potential application in 4D printing. This approach to developing self-healing ferrogels with biocompatible polysaccharides may prove useful in designing and fabricating drug delivery systems and tissue engineering scaffolds, via 3D printing.

Nose-to-Brain Delivery of Cancer-Targeting Paclitaxel-Loaded Nanoparticles Potentiates Antitumor Effects in Malignant Glioblastoma.

Glioblastoma multiforme (GBM) is an aggressive tumor with no curative treatment. The tumor recurrence after resection often requires chemotherapy or radiation to delay the infiltration of tumor remnants. Intracerebral chemotherapies are preferentially being used to prevent tumor regrowth, but treatments remain unsuccessful because of the poor drug distribution in the brain. In this study, we investigated the therapeutic efficacy of cancer-targeting arginyl-glycyl-aspartic tripeptide (RGD) conjugated paclitaxel (PTX)-loaded nanoparticles (NPs) against GBM by nose-to-brain delivery. Our results demonstrated that RGD-modified PTX-loaded NPs showed cancer-specific delivery and enhanced anticancer effects in vivo. The intranasal (IN) inoculation of RGD-PTX-loaded NPs effectively controls the tumor burden (75 ± 12% reduction) by inducing apoptosis and/or inhibiting cancer cell proliferation without affecting the G0 stage of normal brain cells. Our data provide therapeutic evidence supporting the use of intranasally delivered cancer-targeted PTX-loaded NPs for GBM therapy.

Controlling the porous structure of alginate ferrogel for anticancer drug delivery under magnetic stimulation.

Stimulus-responsive drug delivery systems have been widely used for many biomedical applications. Magnetic stimulation may serve as an important external stimulus for drug delivery. In this study, we hypothesized that the on-demand release of anticancer drugs could be achieved with a macroporous alginate ferrogel under the influence of magnetic stimulation to enhance therapeutic efficacy in a tumor-bearing mouse model. A ferrogel containing alginate, iron oxide nanoparticle, and gelatin particle was prepared by ionic crosslinking with calcium ions and dissolving the gelatin particle at 37 °C. We investigated the influence of porosity on the degree of deformation of alginate ferrogel and evaluated the release behavior of doxorubicin (DOX) by applying magnetic field to the ferrogel. In vitro viability of cancer cells cultured with DOX-releasing macroporous alginate ferrogel after magnetic stimulation was greatly decreased compared to that of cells cultured with alginate ferrogel. The therapeutic efficacy of DOX-releasing macroporous alginate ferrogel also increased in tumor-bearing mice following magnetic stimulation. Thus, this approach to design a ferrogel responsive to magnetic stimulation may prove useful for the development of smart drug delivery systems.

Hyaluronate-alginate hybrid hydrogels prepared with various linkers for chondrocyte encapsulation.

Tissue engineering typically requires a use of scaffolds when delivering tissue-specific cells to be engineered. Hydrogels are frequently used as scaffolds, because their composition, structure, and function resemble the natural tissue extracellular matrix. In this study, hyaluronate-alginate hybrid (HAH) was synthesized by conjugating alginate (ALG) with the hyaluronate (HA) backbone using various types of linkers. HAH hydrogel was prepared by physically cross-linking the HAH polymer in the presence of calcium ions without chemical cross-linkers. The mechanical stiffness of HAH hydrogel was significantly affected by changing the type of a linker between HA and ALG. The mechanical stiffness increased with increasing linker length, likely due to enhanced intermolecular reactions between HA and ALG. This enables controlling the mechanical properties of HAH hydrogels. The types of linkers used to synthesize HAHs also influenced the chondrogenic differentiation of ATDC5 cells cultured in HAH hydrogel in vitro. This hybrid system that can change the mechanical stiffness by varying the linker type while maintaining the cross-linking density may be useful to design and fabricate scaffolds for tissue engineering applications, including cartilage regeneration.

Three-Dimensional Bioprinting of Cell-Laden Constructs Using Polysaccharide-Based Self-Healing Hydrogels.

Development of biomaterial-based bioinks is critical for replacement and/or regeneration of tissues and organs by three-dimensional (3D) printing techniques. However, the number of 3D-printable biomaterials in practical use remains limited despite the rapid development of 3D printing techniques. Controlling the flow properties of bioinks and mechanical properties of the resultant printed objects is key considerations in the design of biomaterial-based bioinks for practical applications. In this study, a printable hydrogel comprising biocompatible polysaccharides that has potential for cartilage regeneration via tissue engineering approaches was designed. Self-healing hydrogels were prepared from partially oxidized hyaluronate (OHA) and glycol chitosan (GC) in the presence of adipic acid dihydrazide (ADH). The self-healing ability of OHA/GC/ADH hydrogels was attributed to the combination of two dynamic bonds in the gels, including imine bonds obtained via a Schiff base reaction between OHA and GC, as well as acylhydrazone bonds formed by the reaction between OHA and ADH. The OHA/GC/ADH hydrogels did not require any postgelation or additional cross-linking processes for use in the fabrication of 3D constructs using an extrusion-based 3D printer. The concentrations and molecular weights of the constituent polymers were found to be critical parameters affecting the flow and mechanical properties of the self-healing hydrogels, which showed great potential as bioinks for fabricating cell-laden structures using a 3D printer. The expression of chondrogenic marker genes such as SOX-9 and collagen type II of ATDC5 cells encapsulated in the OHA/GC/ADH hydrogel was not significantly affected by the printing process. This self-healing hydrogel system may have the potential in tissue engineering applications, including cartilage regeneration.

Hyaluronate-alginate hybrid hydrogels modified with biomimetic peptides for controlling the chondrocyte phenotype.

Hyaluronate-based hydrogels have been widely exploited as synthetic extracellular matrices in many tissue engineering applications, including cartilage tissue engineering. Hyaluronate-based hydrogels are typically prepared by chemical cross-linking reactions, in which chemical reagents may induce side effects, unless they are completely removed after the cross-linking reaction. We thus suggest the utilization of hybrid materials composed of hyaluronate as a main chain and alginate for physical cross-linking to simply form hydrogels in the presence of calcium ions under physiological conditions. In this study, we hypothesized that the introduction of biomimetic peptides to hyaluronate-alginate hybrid (HAH) hydrogels could be useful to regulate the chondrocyte phenotype, including chondrogenic differentiation. HAH was modified with the arginine-glycine-aspartate (RGD) peptide as a cell-matrix interaction motif and/or histidine-alanine-valine (HAV) as a cell-cell interaction motif. The HAV peptide is known to bind to cadherin, which is a key factor involved in homophilic cell-cell interactions as well as chondrogenesis. The viability and growth of mouse chondrocytes (ATDC5 cells) increased significantly when cultured on RGD-modified HAH hydrogels. Cell aggregates formed on HAV-modified HAH hydrogels, resulting in enhanced chondrogenic differentiation via enhanced cell-cell interactions by HAV modification. Interestingly, a synergistic effect of HAV and RGD peptides within HAH hydrogels on chondrogenesis was found in 3-D experiments in vitro. This approach to utilizing physically cross-linkable hyaluronate-based hydrogels presenting biomimetic peptides has potential applications in tissue engineering, including cartilage regeneration.