Hyaluronic Acid Polymer Research Articles

Antioxidant Activity of Dopamine-Modified Hydrogels Containing Cross-linked Hyaluronic Acid

Purpose : : In this study, we made dopamine-functionalized hydrogels containing a cross-linked hyaluronic acid (HA) network and investigated their antioxidant activities.Methods : In the first step, we made poly hydroxyethyl methacrylate(p(HEMA))-based hydrogels post-modified with an interpenetrating polymer network(IPN) structure composed of HA polymers and a p(HEMA) network. The subsequent functionalization with dopamine via an amide coupling reaction resulted in the antioxidant hydrogels. Their antioxidant activities were evaluated using 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays.Results : The dopamine-modified hydrogels exhibited significant antioxidant activities, when compared to unmodified control. The presence of the HA–IPN structure improved the surface wettability of the hydrogel while dopamine-conjugated IPN hydrogel did not demonstrate the significant difference compared to hydrogel control. Dopamine-modified hydrogels exhibited high transmittance (>88%).Conclusion : The results demonstrate that the development of antioxidant hydrogels based on dopamine-conjugated HA–IPN structures may help develop ophthalmic and biomedical materials.

The Microfluidic Technique and the Manufacturing of Polysaccharide Nanoparticles.

The microfluidic technique has emerged as a promising tool to accelerate the clinical translation of nanoparticles, and its application affects several aspects, such as the production of nanoparticles and the in vitro characterization in the microenvironment, mimicking in vivo conditions. This review covers the general aspects of the microfluidic technique and its application in several fields, such as the synthesis, recovering, and samples analysis of nanoparticles, and in vitro characterization and their in vivo application. Among these, advantages in the production of polymeric nanoparticles in a well-controlled, reproducible, and high-throughput manner have been highlighted, and detailed descriptions of microfluidic devices broadly used for the synthesis of polysaccharide nanoparticles have been provided. These nanoparticulate systems have drawn attention as drug delivery vehicles over many years; nevertheless, their synthesis using the microfluidic technique is still largely unexplored. This review deals with the use of the microfluidic technique for the synthesis of polysaccharide nanoparticles; evaluating features of the most studied polysaccharide drug carriers, such as chitosan, hyaluronic acid, and alginate polymers. The critical assessment of the most recent research published in literature allows us to assume that microfluidics will play an important role in the discovery and clinical translation of nanoplatforms.

Hyaluronic acid based nanocrystals hydrogels for enhanced topical delivery of drug: A case study

The objective of this study is to use a carbohydrate polymer hyaluronic acid (HLA) as matrix to design novel transdermal nanogel loading poorly soluble drug nanocrystals. Baicalin nanocrystals (BCA-NC) were prepared by coupling homogenization technology and spray-drying technology. The morphology, the rheological behavior and transdermal permeation studies of HLA based BCA-NC-gel were evaluated. The results demonstrated that the BCA-NC could be successfully prepared in terms of trehalose after spray-drying. The trehalose could prevent the aggregation of BCA-NC during spray-drying. It was discovered that, the BCA-NC-gel with 1% HLA possessed the favorable gelatin capacity and thinning shear rheological property. In vitro transdermal permeation studies of BCA-NC-gel/HLA studies indicated a marked increase in the skin permeation of BCA. And the transdermal flux of BCA-NC-gel with 1% HLA were 20.65-fold higher (p < 0.01) than that of coarse BCA-gel, which could be attributed to particles size reduction of BCA-NC and bioadhesive property of HLA. And the morphology characterization of BCA-NC-gel/HLA demonstrated that BCA-NC could be imprisoned into the gel network of HLA, which might prevent it from aggregation in gel. In conclusion, HLA based nanogel system is a promising carrier for effectively transdermal delivery of poorly soluble drug.

PD-1 Blockade for Improving the Antitumor Efficiency of Polymer-Doxorubicin Nanoprodrug.

Chemotherapy is well recognized to induce immune responses during some chemotherapeutic drugs-mediated tumor eradication. Here, a strategy involving blocking programmed cell death protein 1 (PD-1) to enhance the chemotherapeutic effect of a doxorubicin nanoprodrug HA-Psi-DOX is proposed and the synergetic mechanism between them is further studied. The nanoprodrugs are fabricated by conjugating doxorubicin (DOX) to an anionic polymer hyaluronic acid (HA) via a tumor overexpressed matrix metalloproteinase sensitive peptide (CPLGLAGG) for tumor targeting and enzyme-activated drug release. Once accumulated at the tumor site, the nanoprodrug can be activated to release antitumor drug by tumor overexpressed MMP-2. It is found that HA-Psi-DOX nanoparticles can kill tumor cells effectively and initiate an antitumor immune response, leading to the upregulation of interferon-γ. This cytokine promotes the expression of programmed cell death protein-ligand 1 (PD-L1) on tumor cells, which will cause immunosuppression after interacting with PD-1 on the surface of lymphocytes. The results suggest that the therapeutic efficiency of HA-Psi-DOX nanoparticles is significantly improved when combined with checkpoint inhibitors anti-PD-1 antibody (α-PD1) due to the neutralization of immunosuppression by blocking the interaction between PD-L1 and PD-1. This therapeutic system by combining chemotherapy and immunotherapy further increases the link between conventional tumor therapies and immunotherapy.

Viral Capsids Mimicking Based on pH-Sensitive Biodegradable Polymeric Micelles for Efficient Anticancer Drug Delivery.

Bio-inspired supramolecular self-assembly have been widely explored in biomedical engineering, especially in the field of drug delivery. Here, viral capsid analogue pH-Sensitive polymeric micelles HA-Hyd-DOX were reported, where natural polysacarrides hyaluronic acid (HA) and anticancer drug doxorubicin (DOX), were linked through a hydrazone bond with a high drug loading content of 33.09 wt%. The polymeric micelles look like artificial virus capsids from "core-shell" structures. In addition, the polymeric backbone HA and hydrazone bonds were destroyed in the presence of hyaluronidase in cancer cells and under the acidic conditions of pH = 5 respectively, thereby prodrug-based polymeric micelles could penetrate into the tumor and DOX could be released in lysosomes to enhance anticancer efficacy. With the comparison of typical prodrug-based polymeric micelles mPEG-Hyd-DOX system where DOX was linked to methoxy poly(ethylene glycol) with a hydrazone bond linkage, HA-Hyd-DOX showed greater inhibition to cancer cells due to the better penetration. Such viral capsids mimicking polymeric micelles provided some remarkable benefits for drug delivery, including, high drug loading efficiency, controlled drug release and excellent biodegradable.

Characterization of Recombinant Bovine Sperm Hyaluronidase and Identification of an Important Asn-X-Ser/Thr Motif for Its Activity.

Hyaluronidases are a family of enzymes that catalyse the breakdown of hyaluronic acid, which is abundant in the extracellular matrix and cumulus oocyte complex. To investigate the activity of recombinant bovine sperm hyaluronidase 1 (SPAM1) and determine the effect of the Asn-X-Ser/Thr motif on its activity, the bovine SPAM1 open reading frame was cloned into the mammalian expression vector pCXN2 and then transfected to the HEK293 cell line. Expression of recombinant bovine hyaluronidase was estimated using a hyaluronidase activity assay with gel electrophoresis. Recombinant hyaluronidase could resolve highly polymeric hyaluronic acid and also caused dispersal of the cumulus cell layer. Comparative analysis with respect to enzyme activity was carried out for the glycosylated and deglycosylated bovine sperm hyaluronidase by N-glycosidase F treatment. Finally, mutagenesis analysis revealed that among the five potential N-linked glycosylation sites, only three contributed to significant inhibition of hyaluronic activity. Recombinant bovine SPAM1 has hyaluronan degradation and cumulus oocyte complex dispersion ability, and the N-linked oligosaccharides are important for enzyme activity, providing a foundation for the commercialization of hyaluronidase.

Strategy for Preparing Mechanically Strong Hyaluronic Acid–Silica Nanohybrid Hydrogels via In Situ Sol–Gel Process

AbstractA hybridization method to prepare a hyaluronic acid (HA)‐based nanohybrid hydrogel is proposed that introduces an additional inorganic silica network via an in situ sol–gel process. HA hydrogels have been extensively studied because of their excellent biocompatibility and biological functions; however, their poor mechanical strength hinders their use in tissue engineering applications. In the present work, the sol–gel technique is employed to achieve the formation of a structurally organized silica network in the HA hydrogel matrix rather than mixing of discrete particles with the HA polymer matrix. Importantly, the silica densification process results in significant enhancement of the mechanical properties. In addition, the nanohybrid hydrogels exhibit great degradation resistance and bioactivity on both fibroblast and pre‐osteoblast cells. Moreover, the physical characteristics and biological properties can be modulated by varying the silica content; these materials thus show great potential for a wide range of applications for soft and hard tissues.

Uptake and light-induced cytotoxicity of hyaluronic acid-grafted liposomes containing porphyrin in tumor cells

Porphyrins are used in clinic as the photosensitizers for cancer treatment. However, most porphyrins tend to form aggregates in aqueous solution due to their hydrophobicity, which results in significantly reduced photocytotoxicities. Therefore, their applications in clinic are limited. To overcome these disadvantages, drug-carriers are designed for the delivery of porphyrins. In this study, we designed the surface modified liposomes for porphyrin delivery. Four types of porphyrins were loaded in liposome separately. We systemically investigated the porphyrin-containing liposomes in terms of their physicochemical properties as well as light-responsive properties. The hyaluronic acid derivative with aldehyde group was used to modify the liposome-porphyrin via the “click” reaction in order to enhance the uptake in CD44 overexpressed cancer cells. The results showed that 10,15,20-tetrakis(4-hydroxyphenyl) porphyrin (p-OH) had highest loading efficiency. The liposomes with p-OH showed highest toxicity to the cells with or without light exposure. The uptake of hyaluronic acid (HA) coated Lip-p-OH was proved to be specific to MDA-MB-231 cells by pretreating the cells with natural HA polymer as the block agent. This study provided an efficient strategy to enhance the cytotoxicity of porphyrin in aqueous solution, and to increase the affinity of porphyrin to the cancer cells.

Influence of Hyaluronic Acid Transitions in Tumor Microenvironment on Glioblastoma Malignancy and Invasive Behavior.

The extracellular matrix (ECM) is critical in tumor growth and invasive potential of cancer cells. In glioblastoma tumors, some components of the native brain ECM such as hyaluronic acid (HA) have been suggested as key regulators of processes associated with poor patient outlook such as invasion and therapeutic resistance. Given the importance of cell-mediated remodeling during invasion, it is likely that the molecular weight of available HA polymer may strongly influence GBM progression. Biomaterial platforms therefore provide a unique opportunity to systematically examine the influence of the molecular weight distribution of HA on GBM cell activity. Here we report the relationship between the molecular weight of matrix-bound HA within a methacrylamidefunctionalized gelatin (GelMA) hydrogel, the invasive phenotype of a patient-derived xenograft GBM population that exhibits significant in vivo invasivity, and the local production of soluble HA during GBM cell invasion. Hyaluronic acid of different molecular weights spanning a range associated with cell-mediated remodeling (10, 60, and 500 kDa) was photopolymerized into GelMA hydrogels, with cell activity compared to GelMA only conditions (-HA). Polymerization conditions were tuned to create a homologous series of GelMA hydrogels with conserved poroelastic properties (i.e., shear modulus, Poisson's ratio, and diffusivity). GBM migration was strongly influenced by HA molecular weight; while markers associated with active remodeling of HA (hyaluronan synthase and hyaluronidase) were found to be uninfluenced. These results provide new information regarding the importance of local hyaluronic acid content on the invasive phenotype of GBM.

Metformin-Loaded Hyaluronic Acid Nanostructure for Oral Delivery.

The objective of this study was to develop a nanodelivery system containing a mucoadhesive polymer hyaluronic acid (HA) for oral delivery. Metformin was used as a model drug. Blank and drug-loaded HA nanostructures were prepared by precipitation method and characterized for particle size (PS), zeta potential (ZP), physical stability (over 65days), surface morphology, moisture content, and physical state of the drug in the nanostructures. The cytotoxicity and hemolysis potential of the delivery system was assessed in Caco-2 cells and whole human blood, respectively. The in vitro release of metformin and its uptake in Caco-2 cells was evaluated using high-performance liquid chromatography. Ex vivo permeability of metformin was measured through goat intestinal membrane. The nanoparticles were physically stable and neutrally charged with an average PS of 114.53 ± 12.01nm. This nanodelivery system existed as nanofibers containing metformin in a crystalline state. This delivery system released the drug rapidly with > 50% of metformin released within 1h. Cellular uptake studies on Caco-2 cells indicated higher uptake of metformin from nanoparticle as compared to metformin in solution, up to first 45min. Ex vivo permeability studies on the other hand showed a higher metformin permeability from solution relative to that from nanoparticles through the goat intestinal membrane. Metformin nanoparticles were non-toxic at therapeutic concentrations in Caco-2 cells and showed no hemolytic effect to RBCs. This study indicates the preparation, characterization, as well as the potential use of HA nanostructures for oral delivery.

Intermolecular Interactions between Bottlebrush Polymers Boost the Protection of Surfaces against Frictional Wear

Polymers exhibiting the bottlebrush (BB) architecture have excellent lubricating properties. However, to motivate their use in real life systems, they must also protect surfaces against frictional damage. In this article, we synthesized a library of polyzwiterrionic bottlebrush polymers of different architectures to explore the effect of intermolecular interactions on their conformation at interfaces and their tribological properties. Using the surface forces apparatus, we show that increasing the number of adhesive blocks on the BB polymers does not impact the friction coefficient on mica surfaces, μ, which remained close to μ = 0.02 but drastically increased the threshold pressure, P*, at which wear initiates from P* = 0.4 ± 0.1 up to 8.0 ± 0.8 MPa. In mixtures of high molecular weight hyaluronic acid and BB polymers, a synergistic interaction between polymers occurred, leading to a significant increase in P*, independently of the BB polymer tested and even reaching superprotection for strongly interact...

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High-Molecular-Weight Hyaluronic Acid.

Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial HA production, the potential of protein engineering has barely been realised. Herein, knowledge-gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain-length specificity and a twofold increase in mass-based turnover number. Seven improved pmHAS variants out of 1392 generated by error-prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell-free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N-terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.

MnO2 Motor: A Prospective Cancer-Starving Therapy Promoter.

Here, a tumor-targeted MnO2 motor nanosystem (designed as MG/HA) was constructed by the assembly of glucose oxidase (GOD), manganese dioxide (MnO2), and glycoprotein CD44-targeting polymer hyaluronic acid (HA) to elevate cancer-starving therapy efficacy in solid tumor. Upon the specific uptake of MG/HA by CD44 overexpressed cancer cells, GOD catalyzed the oxidation of glucose into gluconic acid and hydrogen peroxide (H2O2) accompanying the consumption of oxygen (O2). Meanwhile, MnO2 would react with H2O2 and acid to generate O2, which is in turn supplied to the glucose-depletion process, running like a loop. As a result, MnO2 is displayed as a motor to promote the rate of glucose depletion that contributed to the starving therapy. In contrast to G/HA, MG/HA could not only achieve effective glucose consumption to depress cancer progression, but also alleviate hypoxia and reduce the expression of Glut1 to inhibit the metabolism for further restraining the tumor aggressiveness and metastasis. The concept of MnO2 motor shows a promising prospect to overcome the restriction of the starving therapy.

Natural polymer-based hydrogels for cartilage regeneration

Purpose: Cartilage is a tough, flexible tissue found throughout the body. It can become damaged because of a sudden injury, such as a sports injury, or gradual wear and tear (osteoarthritis). Injured articular cartilage has a poor regenerative capability, for this reason, tissue engineering may provide a fundamental therapeutic solution to repair cartilage injury. Human articular cartilages are composed of dense extracellular matrix and chondrocytes. One of the major components in the extracellular matrix is hyaluronic acid. The combination of hyaluronic acid with other natural polymers to prepare hydrogel scaffolds may be potentially useful in many tissue engineering applications, including cartilage regeneration. We propose the synthesis of hydrogels based on hyaluronic acid and chitosan by a new strategy of synthesis using di-isocyanates to obtain an interpenetrated network of chitosan and hyaluronic acid. Methods: Different kinds of hydrogels were synthesized using hyaluronic acid (Bioibérica, Spain), natural polymers (gelatine, chitosan) and stabilized with diisocianates. The hydrogels were charged with chondroitin sulfate (Bioibérica, Spain). Morphological Characterization: The hydrogels were characterized by scanning electron microscopy and energy dispersive X-ray microscopy in order to study the morphology and the composition of the hydrogels. Hydrogel Swelling: Dynamic swelling experiments were performed by placing discs of stabilized hydrogels in PBS at 37.0 °C, and measuring their weight gain as a function of time. Biocompatibility: Cytotoxicity in vitro assays (MTT) and cell adhesion tests (Alamar Blue) were performed on the hydrogels using different cell lines to corroborate the biocompatibility of the hydrogels. Results: All the synthesized hydrogels present a high interconnected porosity. The studied hydrogels swell around 200%. Aaround five hours equilibrium is reached and this equilibrium depend on the ratio hyaluronic acid/natural polymer increasing with the amount of hyaluronic acid present on the hydrogel. MTT assay indicated that the extracts collected at different times from the hydrogels are not cytotoxic and could be considered biocompatible. Cell adhesion assay indicates that the hydrogels support cell adhesion and proliferation, and this behaviour is better on SC-loaded hydrogels. Conclusions: Hydrogels based on hyaluronic acid and natural polymers are proposed as scaffolds for tissue engineering. The presence of interconnected porous and the good swelling properties and biocompatibility represent a great advantage for application in cartilage regeneration. The addition of drugs or glycosaminoglycan is being studied to improve the cartilage regeneration.

Super Liquid Crystalline Polysaccharides Produced by Ultimately-ecological Microreactors

Cyanobacteria fix carbon dioxide and nitrogen from the atmosphere using solar energy to produce various biomolecules, and thus are regarded as ultimately ecological microreactors. Sacran is a cyanobacterial polysaccharide with a very high molecular weight of 29 Mg/mol, which is extracted from Aphanothece sacrum cyanobacterium mass-cultivated in freshwater environments such as river or spring. Sacran is a water-soluble heteropolysaccharide comprising more than 6 kinds of sugar residues and contains 12% sulfate anionic groups and 27% carboxyls. Sacran has a super-absorbent function of water, which can retain 6000 mL for 1 g specimen, due to very long hydrating chains. The value is much higher than hyaluronic acid or conventional super-absorbent polymers. Sacran exhibits self-orienting behavior in dilute solution at a concentration range over 0.25 wt%, which is quite low when compared with conventional liquid crystalline polysaccharides. Mesogenic helical chains of sacrans have extremely high aspect ratios of 1600 for highly persistent lengths of 32 micrometer. Through the liquid crystallinity, sacran solution shows a shear-thinning behavior and the solution spread over a substrate such as biological skin very efficiently to create a thin layer. Applied on atopic dermatitis skin sacran solution exerts excellent moisturizing effect and anti-itching action.

Amplified polarization properties of electrospun nanofibers containing fluorescent dyes and helical polymer.

Nanofibers with the cationic fluorescent molecules, 1-pyrenemethylamine hydrochloride (PyrMA) and α,β,γ,δ-tetrakis(1-methylpyridinium-4-yl)porphyrin p-toluenesulfonate (TMPyP), in the presence of complementary anionic chiral polysaccharide, hyaluronic acid (HA) and transparent polymer, polyethylene oxide (PEO) were successfully prepared using an electrospinning technique. The results showed that the fluorescence spectra of electrospun nanofibers (ENFs) of PEO-HA-PyrMA exhibit a higher amount of pyrene excimer emission when the [PyrMA]/[HA] ratio was increased from 0.5 to 1, while the concentration of TMPyP had no remarkable effect on the fluorescence of the PEO-HA-TMPyP ENFs. From circular dichroism (CD) characterization, the ENFs of both dyes exhibit stronger intensities in the apparent CD spectra upon increasing the dye ratio. Moreover, they also showed higher CD intensities when compared with those of solutions and thin films prepared using other methods. The apparent CD in this work was proved to mainly originate from linear dichroism (LD) and linear birefringence (LB) obtained by macroscopic alignment along the electrospinning process. These results suggest the advantages of molecular orientation via electrospinning, together with chirality induction by incorporation of a chiral HA polymer. The enhancement of these polarization properties provides great possibilities for improving fluorescent-chiral nanofibers for further optoelectronic applications.

Intermolecular G-Quadruplex Induces Hyaluronic Acid-DNA Superpolymers Causing Cancer Cell Swelling, Blebbing, and Death.

Over the past decade, nanomedicine has gained considerable attraction through its relevance, for example, in "smart" delivery, thus creating platforms for novel treatments. Here, we report a natural polymer-DNA conjugate that undergoes self-assembly in a K+-dependent fashion to form a G-quadruplex (GQ) and generate superpolymeric structures. We derivatized a thiolated conjugate of the naturally occurring glycosaminoglycan polymer hyaluronic acid (HASH) with short G-rich DNA (HASH-DNA) that can form an intermolecular noncanonical GQ structure. Gel mobility shift assay and circular dichroism measurements confirmed HASH conjugation to DNA and K+-dependent GQ formation, respectively. Transmission electron microscopy and scanning electron microscopy results indicated that the addition of K+ to the HASH-DNA conjugate led to the formation of micron-range structures, whereas control samples remained unordered and as a nebulous globular form. Confocal microscopy of a fluorescently labeled form of the superpolymer verified increased cellular uptake. The HASH-DNA conjugates showed toxicity in HeLa cells, whereas a scrambled DNA (Mut) conjugate HASH-Mut showed no cytotoxicity, presumably because of nonformation of the superpolymeric structure. To understand the mechanism of cell death and if the superpolymeric structure is responsible for it, we monitored the cell size and observed an average of 23% increase in size compared to 4.5% in control cells at 4.5 h. We believe that cellular stress is generated presumably by the intracellular assembly of this large superpolymeric nanostructure causing cell blebbing with no exit option. This approach provides a new strategy of cellular delivery of a targeted naturally occurring polymer and a novel way to induce superpolymeric structure formation that acts as a therapeutic.

Comparison of Compressive Stress-Relaxation Behavior in Osteoarthritic (ICRS Graded) Human Articular Cartilage.

Osteoarthritis (OA) is a common joint disorder found mostly in elderly people. The role of mechanical behavior in the progression of OA is complex and remains unclear. The stress-relaxation behavior of human articular cartilage in clinically defined osteoarthritic stages may have importance in diagnosis and prognosis of OA. In this study we investigated differences in the biomechanical responses among human cartilage of ICRS grades I, II and III using polymer dynamics theory. We collected 24 explants of human articular cartilage (eight each of ICRS grade I, II and III) and acquired stress-relaxation data applying a continuous load on the articular surface of each cartilage explant for 1180 s. We observed a significant decrease in Young’s modulus, stress-relaxation time, and stretching exponent in advanced stages of OA (ICRS grade III). The stretch exponential model speculated that significant loss in hyaluronic acid polymer might be the reason for the loss of proteoglycan in advanced OA. This work encourages further biomechanical modelling of osteoarthritic cartilage utilizing these data as input parameters to enhance the fidelity of computational models aimed at revealing how mechanical behaviors play a role in pathogenesis of OA.

Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells.

Growth factors hold great promise for regenerative therapies. However, their clinical use has been halted by poor efficacy and rapid clearance from tissue, necessitating the delivery of extremely high doses to achieve clinical effectiveness which has raised safety concerns. Thus, strategies to either enhance growth factor activity at low doses or to increase their residence time within target tissues are necessary for clinical success. In this study, we generated multivalent conjugates (MVCs) of basic fibroblast growth factor (bFGF), a key growth factor involved in angiogenesis and wound healing, to hyaluronic acid (HyA) polymer chains. Multivalent bFGF conjugates (mvbFGF) were fabricated with minimal non-specific interaction observed between bFGF and the HyA chain. The hydrodynamic radii of mvbFGF ranged from ∼50 to ∼75 nm for conjugation ratios of bFGF to HyA chains at low (10 : 1) and high (30 : 1) feed ratios, respectively. The mvbFGF demonstrated enhanced bioactivity compared to unconjugated bFGF in assays of cell proliferation and migration, processes critical to angiogenesis and tissue regeneration. The 30 : 1 mvbFGF outperformed the 10 : 1 conjugate, which could be due to either FGF receptor clustering or interference with receptor mediated internalization and signal deactivation. This study simultaneously investigated the role of both protein to polymer ratio and multivalent conjugate size on their bioactivity, and determined that increasing the protein-to-polymer ratio and conjugate size resulted in greater cell bioactivity.

Hyaluronic Acid-Based pH-Sensitive Polymer-Modified Liposomes for Cell-Specific Intracellular Drug Delivery Systems.

For the enhancement of therapeutic effects and reduction of side effects derived from anticancer drugs in cancer chemotherapy, it is imperative to develop drug delivery systems with cancer-specificity and controlled release function inside cancer cells. pH-sensitive liposomes are useful as an intracellular drug delivery system because of their abilities to transfer their contents into the cell interior through fusion or destabilization of endosome, which has weakly acidic environment. We earlier reported liposomes modified with various types of pH-sensitive polymers based on synthetic polymers and biopolymers as vehicles for intracellular drug delivery systems. In this study, hyaluronic acid (HA)-based pH-sensitive polymers were designed as multifunctional polymers having not only pH-sensitivity but also targeting properties to cells expressing CD44, which is known as a cancer cell surface marker. Carboxyl group-introduced HA derivatives of two types, MGlu-HA and CHex-HA, which have a more hydrophobic side chain structure than that of MGlu-HA, were synthesized by reaction with various dicarboxylic anhydrides. These polymer-modified liposomes were stable at neutral pH, but showed content release under weakly acidic conditions. CHex-HA-modified liposomes delivered their contents into CD44-expressing cells more efficiently than HA-modified or MGlu-HA-modified liposomes or unmodified liposomes, whereas the same liposomes were taken up only slightly by cells expressing CD44 proteins less. Competition assay using free HA or other polymers revealed that HA derivative-modified liposomes might be recognized by CD44. Therefore, HA-derivative-modified liposomes are useful as cell-specific intracellular drug delivery systems.