Free Hyaluronic Acid Research Articles

Freeze Drying of Polymer Nanoparticles and Liposomes Exploiting Different Saccharide-Based Approaches.

Biodegradable nanocarriers represent promising tools for controlled drug delivery. However, one major drawback related to their use is the long-term stability, which is largely influenced by the presence of water in the formulations, so to solve this problem, freeze-drying with cryoprotectants has been proposed. In the present study, the influence of the freeze-drying procedure on the storage stability of poly(lactide-co-glycolide) (PLGA) nanoparticles and liposomes was evaluated. In particular, conventional cryoprotectants were added to PLGA nanoparticle and liposome formulations in various conditions. Additionally, hyaluronic acid (HA), known for its ability to target the CD44 receptor, was assessed as a cryoprotective excipient: it was added to the nanocarriers as either a free molecule or conjugated to a phospholipid to increase the interaction with the polymer or lipid matrix while exposing HA on the nanocarrier surface. The formulations were resuspended and characterized for size, polydispersity index, zeta potential and morphology. It was demonstrated that only the highest percentages of cryoprotectants allowed the resuspension of stable nanocarriers. Moreover, unlike free HA, HA-phospholipid conjugates were able to maintain the particle mean size after the reconstitution of lyophilized nanoparticles and liposomes. This study paves the way for the use of HA-phospholipids to achieve, at the same time, nanocarrier cryoprotection and active targeting.

Self-assembly based aerosolized hyaluronic acid (HA) loaded niosomes for lung delivery: An in-vitro and in-vivo evaluation

Hyaluronic acid, a biological macromolecule, was utilized as a drug for loading into niosomes. The ethanol injection method was used to formulate the niosomes using Cholesterol as the lipid and Span 80 as the non-ionic surfactant. HA-loaded niosomes showed a particle size of 177.6 nm and a % Entrapment Efficiency (%EE) of >95%. Drug release studies from the HA-loaded niosomes showed a controlled release profile for up to 4 days. Robust stability of the HA-loaded niosomes at both R.T (25–30 °C) and 4–8 °C was obtained. DPPH (2,2-diphenyl-1-picrylhydrazyl) antioxidant assay exhibited a greater enzyme inhibition than the free HA solution. In-vivo pharmacokinetic drug estimation parameters revealed a greater HA plasma concentration than free HA solution through the aerosol route. Moreover, organ biodistribution studies showed a greater localization of HA in lungs than in other organs.

CD44 Receptor-Mediated/Reactive Oxygen Species-Sensitive Delivery of Nanophotosensitizers against Cervical Cancer Cells.

Stimulus-sensitive, nanomedicine-based photosensitizer delivery has an opportunity to target tumor tissues since oxidative stress and the expression of molecular proteins, such as CD44 receptors, are elevated in the tumor microenvironment. The aim of this study is to investigate the CD44 receptor- and reactive oxygen species (ROS)-sensitive delivery of nanophotosensitizers of chlorin e6 (Ce6)-conjugated hyaluronic acid (HA) against HeLa human cervical cancer cells. For the synthesis of nanophotosensitizers, thioketal diamine was conjugated with the carboxyl group in HA and then the amine end group of HA-thioketal amine conjugates was conjugated again with Ce6 (Abbreviated as HAthCe6). The HAthCe6 nanophotosensitizers were of small diameter, with sizes less than 200. Their morphology was round-shaped in the observations using a transmission electron microscope (TEM). The HAthCe6 nanophotosensitizers responded to oxidative stress-induced changes in size distribution when H2O2 was added to the nanophotosensitizer aqueous solution, i.e., their monomodal distribution pattern at 0 mM H2O2 was changed to dual- and/or multi-modal distribution patterns at higher concentrations of H2O2. Furthermore, the oxidative stress induced by the H2O2 addition contributed to the disintegration of HAthCe6 nanophotosensitizers in morphology, and this phenomenon accelerated the release rate of Ce6 from nanophotosensitizers. In a cell culture study using HeLa cells, nanophotosensitizers increased Ce6 uptake ratio, ROS generation and PDT efficacy compared to free Ce6. Since HA specifically bonds with the CD44 receptor of cancer cells, the pretreatment of free HA against HeLa cells decreased the Ce6 uptake ratio, ROS generation and PDT efficacy of HAthCe6 nanophotosensitizers. These results indicated that intracellular delivery of HAthCe6 nanophotosensitizers can be controlled by the CD44 receptor-mediated pathway. Furthermore, these phenomena induced CD44 receptor-controllable ROS generation and PDT efficacy by HAthCe6 nanophotosensitizers. During in vivo tumor imaging using HeLa cells, nanophotosensitizer administration showed that the fluorescence intensity of tumor tissues was relatively higher than that of other organs. When free HA was pretreated, the fluorescence intensity of tumor tissue was relatively lower than those of other organs, indicating that HAthCe6 nanophotosensitizers have CD44 receptor sensitivity and that they can be delivered by receptor-specific manner. We suggest that HAthCe6 nanophotosensitizers are promising candidates for PDT in cervical cancer.

Hyaluronidase and pH Dual-Responsive Nanoparticles for Targeted Breast Cancer Stem Cells.

pH-responsive and CD44 receptor-mediated targeted nanoparticles for eliminating cancer stem cells (CSCs) were developed based on complexes of PEG-poly(β-amino ester) (PEG-PBAE) micelles (PPM) coated with hyaluronic acid (HA) (HA-coated PPM complex, or HPPMc). Thioridazine (Thz) was loaded into HPPMc with a decent drug loading content. The release results of the drug in vitro showed that Thz was released from the HPPMc, which was stimulated by both the acidic pH and specific enzymes. Cytotoxicity studies on mammospheres (MS) revealed that the toxicity potential of Thz-loaded HPPMc (Thz–HPPMc) at pH 5.5 was better than drug solutions. Compared with that at pH 7.4, a higher cellular uptake of a coumarin-6 (C6)-labeled complex at pH 5.5 was observed, which demonstrated that complexes were efficiently taken up in MS. Meanwhile, free HA competitively inhibited the cellular uptake of HPPMc, which revealed that the uptake mechanism was CD44 receptor-mediated endocytosis. Within the acidic endolysosomal environment, the protonation of PBAE facilitated the escape of the complex from the lysosome and releases the drug. The results of in vivo distribution studies and tumor suppression experiments showed that HPMMc could stay in the tumor site of BALB/c nude mice for a longer period of time, and Thz–HPPMc could significantly improve the tumor-suppressing effect. All these results demonstrated the great potential of the multifunctional nanoparticle system for eliminating CSCs.

Redox sensitive nano-capsules self-assembled from hyaluronic acid-hydroxychloroquine conjugates for CD44-targeted delivery of hydroxychloroquine to combat breast cancer metastasis in vitro and in vivo

Identifying an efficient tumor-targeted drug delivery system is an urgent task for the treatment of metastatic breast cancer. Herein, we report for the first time that a pH and redox dual-responsive polymer prodrug displays prominent inhibition of breast cancer metastasis to the lung. The polymer molecule was synthesized from hyaluronic acid (HA) and hydroxychloroquine (HCQ) through disulfide bonds as redox-sensitive linkers that self-assemble into nanocapsules (HA-ss-HCQ nanocapsules) at pH 7.4. Due to the pKa value of HCQ and built-in disulfide bonds, the nanocapsules were endowed with pH and redox dual-responsive properties. The in vitro drug release curve revealed that HA-ss-HCQ nanocapsules release drugs more quickly under low pH and high redox conditions. Moreover, the nanocapsules displayed active targeting and selectivity to metastatic breast cancer cells. Cellular uptake of Nile red-loaded nanocapsules observed by fluorescence microscopy showed that the nanocapsules exerted significantly enhanced cellular internalization capacity, which was greatly diminished by free HA. The IC50 of HA-ss-HCQ nanocapsules in 4T1 cells was 2.23-fold lower than that of free HCQ. Importantly, wound healing assays and Transwell experiments demonstrated that HA-ss-HCQ nanocapsules greatly inhibited the migration and invasion of 4T1 cells. In particular, the metastasis of 4T1 cells to the lung was also remarkably suppressed by HA-ss-HCQ nanocapsules with minimal toxicity in an in vitro lung metastasis model, which was verified by the detection of macroscopic metastatic nodules and histological examination. In summary, this study provides a promising strategy for active-targeting therapy in metastatic breast cancer.

Hyaluronic Acid in the Intestinal Tract: Influence of Structure, Rheology, and Mucoadhesion on the Intestinal Uptake in Rats.

Oral hyaluronic acid (HA) is a ubiquitous biopolymer that has gained attention as a treatment for local or systemic diseases. Here, we prepared and characterized structures of free HA (f-HA) with a high (>105 Da), intermediate (≤105 Da), and low (≤104 Da) average molar mass (MM); nanoparticles crosslinked with adipic dihydrazide (n-HA); and mixed formulations (mixed-HA) containing f-HA and n-HA. MM distribution determined the structure, hydrodynamic diameter, and zeta potential of the f-HAs. Crosslinking changed the physicochemical properties in n-HA. In vitro tack adhesion assays, using mucin tablets or a viable rat intestinal mucosa, showed better mucoadhesion with f-HA (intermediate MM) and mixed-HA (25% n-HA), especially in the jejunum segment. High MM f-HA presented negligible mucoadhesion. n-HA showed the deepest diffusion into the porous of the membranes. In vivo results showed that, except for high MM f-HA, there is an inverse relationship between rheological changes in the intestinal membrane macerates resulting from mucoadhesion and the effective intestinal permeability that led to blood clearance of the structures. We conclude that the n-HA formulations are promising for targeting other tissues, while formulations of f-HA (intermediate MM) and mixed-HA are better for treating dysbiosis.

The Role of Hyaluronic Acid in Cartilage Boundary Lubrication.

Hydration lubrication has emerged as a new paradigm for lubrication in aqueous and biological media, accounting especially for the extremely low friction (friction coefficients down to 0.001) of articular cartilage lubrication in joints. Among the ensemble of molecules acting in the joint, phosphatidylcholine (PC) lipids have been proposed as the key molecules forming, in a complex with other molecules including hyaluronic acid (HA), a robust layer on the outer surface of the cartilage. HA, ubiquitous in synovial joints, is not in itself a good boundary lubricant, but binds the PC lipids at the cartilage surface; these, in turn, massively reduce the friction via hydration lubrication at their exposed, highly hydrated phosphocholine headgroups. An important unresolved issue in this scenario is why the free HA molecules in the synovial fluid do not suppress the lubricity by adsorbing simultaneously to the opposing lipid layers, i.e., forming an adhesive, dissipative bridge between them, as they slide past each other during joint articulation. To address this question, we directly examined the friction between two hydrogenated soy PC (HSPC) lipid layers (in the form of liposomes) immersed in HA solution or two palmitoyl–oleoyl PC (POPC) lipid layers across HA–POPC solution using a surface force balance (SFB). The results show, clearly and surprisingly, that HA addition does not affect the outstanding lubrication provided by the PC lipid layers. A possible mechanism indicated by our data that may account for this is that multiple lipid layers form on each cartilage surface, so that the slip plane may move from the midplane between the opposing surfaces, which is bridged by the HA, to an HA-free interface within a multilayer, where hydration lubrication is freely active. Another possibility suggested by our model experiments is that lipids in synovial fluid may complex with HA, thereby inhibiting the HA molecules from adhering to the lipids on the cartilage surfaces.

Acetylated Hyaluronic Acid-Poly(L-lactic acid) Conjugate Nanoparticles for Inhibition of Doxorubicinol Production from Doxorubicin

In this study, doxorubicin (DOX)-induced cardiotoxicity was overcome using cancer-recognizable DOX-loaded acetylated hyaluronic aicd-poly(L-lactic acid) (AcHA-PLLA) nanoparticles (DHPs). AcHA-PLLA was synthesized via ring opening polymerization of L-lactide with AcHA as a macroinitiator and characterized by 1H NMR and critical micelle concentration. DHPs were formed by self-assembly via dialysis, were estimated to be 160.7 ± 2.7 nm in average diameter, exhibited a slow rate release profile, and prevented DOX from being degraded in the circulation. In addition, DHPs demonstrated efficient cellular uptake into a colorectal cancer cell line (HCT-116) by CD44 receptor-mediated endocytosis, which was confirmed by in vitro cellular uptake, the competitive inhibition assay with free HA and flow cytometry. Furthermore, DHPs exhibited therapeutic efficacy against HCT-116 cells through receptor-mediated endocytosis: DHPs with free hyaluronic acid (HA) demonstrated 2-fold higher cell viability than DHPs without free HA. DHPs also inhibited doxorubicinol production, which induces cardiotoxicity, from DOX in vivo. The area-under-the-curve (AUC) of DOX administered as free DOX was 109.0 ± 25.0 µg min mL−1 and that from DHPs was 97.2 ± 10.2 µg min mL−1. Although these are comparable, the AUC of doxorubicinol was 1.36 ± 0.18 µg min mL−1 and 0.45 ± 0.12 µg min mL−1, with the treatment of free DOX-HCl and DHPs, respectively, demonstrating the reduction of the metabolic conversion of DOX to doxorubicinol in plasma. These results indicate the role of DHPs in preventing DOX transformation into doxorubicinol, which may contribute to the reduced toxicity. DHPs are promising cancer therapeutic nanoparticles, because they are highly biocompatible and tumor-recognizable, and prevent the DOX transformation..

Abstract 1521: Tumor-produced hyaluronan contributes to the formation tolerogenic immunosuppressive microenvironment

Abstract Immunosuppressive ligand PD-L1 considered as a major player in mechanism of negative regulation of anti-tumor immune response and its expression is highly up-regulated in human and experimental tumors, including bladder cancer. Bladder cancer is characterized by aberrant hyaluronan metabolism resulting in increased HA production in tumor tissue. Hyaluronan or hyaluronic acid (HA) is a prominent component of tumor stroma/microenvironment. Membrane-bound or free extracellular HA favors tumor progression by inducing tumor cell motility, invasive properties, proliferation, production of growth factors and epithelial-mesenchymal transition. Here, we provide evidence that tumor-produced hyaluronan, one of the key tumor stroma components, contributes to the formation of tolerogenic/immunosuppressive tumor microenvironment and stimulated PD-L1 expression. We found that tumor-derived HA specifically binds to CD44 receptor expressed by myeloid precursors, such as myeloid-derived suppressor cells (MDSCs) and promotes its expansion with differentiation toward PD-L1-expressing F4/80+ macrophages. Inhibition of HA synthesis in tumor cells with pharmacologic inhibitor 4-MU, or blockade of CD44 signaling in myeloid cells with antagonistic anti-CD44 antibody, prevented the macrophage differentiation and tumor-induced up-regulation of PD-L1 expression. We also found that tumor-derived HA stimulated production of immunosuppressive and inflammatory factors IL-6, IL-10, TNF-alpha, IL-1beta and PGE2 by myeloid cells in CD44-dependent manner. Moreover, tumor-produced HA stimulated development of PD-L1-expressing macrophages in both murine and human myeloid cells. Our study reveals that tumors may evade the immune system by creating a protective tolerogenic “shield” in the form of tumor-produced HA, which binds to the CD44-expressing tumor-recruited MDSCs, stimulating production of immunosuppressive factors and promoting development of the PD-L1+ macrophages. Inhibiting of hyaluronan synthesis by tumors or targeting HA-CD44 signaling could provide an attractive approach to break tumor-induced immune tolerance and unleash the anti-tumor immune response. Citation Format: Paul R. Dominguez-Gutierrez, Paul Crispen, Sergei A. Kusmartsev. Tumor-produced hyaluronan contributes to the formation tolerogenic immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1521.

One step treatment of talus osteochondral lesions with microfracture and cell free hyaluronic acid based scaffold combination

ObjectiveThe aim of this study was to assess the effectiveness of microfracture and cell free hyaluronic acid (HA) based scaffold combination in the treatment of talus osteochondral defects (OCD). MethodsThis study retrospectively evaluated the clinical results of the 20 patients (14 males and 6 females, mean age at the time of surgery: 32.9 years (range: 16–52 years)) who were treated with MFx and cell-free HA-based scaffold combination for talus OCD smaller than 1.5 cm2 and deeper than 7 mm. Results were evaluated with AOFAS and VAS scores. Also, patients' satisfaction was questioned. ResultsPatients were evaluated after an average follow-up of 20.3 months. Intraoperative measurements showed that mean depth of the lesions were 10.4 ± 1.9 mm after debridement. The mean preoperative AOFAS score was 57.45 ± 9.37, which increased to 92.45 ± 8.4 postoperatively (p < 0.05). VAS score was improved from 7.05 ± 2.45 to 1.65 ± 2.20 postoperatively (p < 0.05). ConclusionMFx and cell-free HA-based scaffold combination appear to be a safe and efficient technique that provide good clinical outcomes for lesions deeper than 7 mm. Level of evidenceLevel IV, Therapeutic Study.

Redox and CD44 Dual‐Responsive Nanophotosensitizer Composed of Chlorin e6‐Conjugated Hyaluronic Acid via Disulfide Linkage for Targeted Photodynamic Treatment of Cancer Cells

For redox‐ and CD44 receptor‐sensitive photodynamic treatment of cancer cells, chlorin e6 (Ce6) was conjugated to the end of hyaluronic acid (HA) via disulfide linkages. Ce6‐conjuated HA (HAssCe6) nanophosensitizers showed small particle size less than 100 nm and spherical morphology. Ce6 was liberated from HAssCe6 nanophotosensitizer with redox‐sensitive manner and Ce6 release rate was faster in the presence of GSH than normal condition. HAssCe6 nanophotosensitizers showed extremely higher Ce6 uptake ratio than Ce6 itself. Pretreatment of HA properly inhibited intracellular uptake of HAssCe6 nanophotosensitizers, indicating that nanophotosensitizers can be entered into cells by CD44 receptor‐mediated pathway. HAssCe6 nanophotosensitizer treatment increased reactive oxygen species production and PDT efficacy with negligible dark toxicity against HCT116 cells. Furthoremore, HAssCe6 nanophotosensitizers was efficiently delivered to tumor tissues of HCT116‐bearing mice while pretreatment of free HA properly inhibited delivery of nanophotosensitizers against tumor tissues. These results indicated that HAssCe6 nanophotosensitizers have redox‐ and CD44‐senstivity for cancer targeting. We suggested HAssCe6 nanophotosensitizers as a promising candidate for PDT of cancer cells.

PH- and GSH-Sensitive Hyaluronic Acid-MP Conjugate Micelles for Intracellular Delivery of Doxorubicin to Colon Cancer Cells and Cancer Stem Cells.

A dual-sensitive polymeric drug conjugate (HA-SS-MP) was synthesized by conjugating hydrophobic 6-mercaptopurine (MP) to thiolated hyaluronic acid (HA) as the carrier and ligand to deliver doxorubicin (Dox) to parental colon cancer and colon cancer stem cells. Because of the amphiphilic nature of HA-SS-MP, it was self-assembled in the aqueous media, and Dox was physically encapsulated in the core of the micelles. The particle size and the zeta potential of the micelle were analyzed by dynamic light scattering (DLS), and the morphology of the micelle was investigated using transmission electron microscopy (TEM). Drug release study results revealed more drug release at pH 5.0 in the presence of GSH than that at the physiological pH value. The cytotoxicity of free Dox was slightly greater than that of Dox-loaded HA-SS-MP micelles. In vitro cytotoxicity of HA-SS-MP and Dox-loaded HA-SS-MP micelles was greater for cancer stem cells (HCT116-CSCs) than for parental HCT116 colon cancer cells and L929 normal fibroblast cells. The MTT and flow cytometry results confirmed that free HA competitively inhibited Dox-loaded HA-SS-MP uptake. Similarly, flow cytometry results revealed anti-CD44 antibody competitively inhibited cellular uptake of Rhodamine B isothiocyanate conjugated micelles, which confirms that the synthesized micelle is uptaken via CD44 receptor. Cell cycle analysis revealed that free drugs and Dox-loaded HA-SS-MP arrested parental HCT116 colon cancer cells at the S phase, while cell arrest was observed at the G0G1 phase in HCT116-CSCs. In addition, ex vivo biodistribution study showed that Dox-loaded HA-SS-MP micelles were accumulated more in the tumor region than in any other organ. Furthermore, the in vivo results revealed that Dox-loaded HA-SS-MP micelles exhibited more therapeutic efficacy than the free drugs in inhibiting tumor growth in BALB/C nude mice. Overall, the results suggested that the synthesized micelles could be promising as a stimuli carrier and ligand for delivering Dox to colon cancer cells and also to eradicate colon cancer stem cells.

A new strategy for the passive skin delivery of nanoparticulate, high molecular weight hyaluronic acid prepared by a polyion complex method

Restoring hyaluronic acid (HA) content is important for maintaining the function of photo-aged skin. This study aimed to evaluate the passive delivery into skin of HA nanoparticles formed by the polyion complex method. Nanoparticles were prepared by mixing and stirring anionic HA with a cationic polymer, protamine, at the charge ratio 55:45. The permeation of fluorescently-labelled HA nanoparticles (HANP) or free HA through hairless mouse skin was characterized in vitro. HANP or free HA was applied to ultraviolet (UV)-irradiated mice in vivo, and their transepidermal water loss (TEWL) was measured after 4 days. HA that had been delivered into skin was separated and characterized by molecular sieve chromatography. HANP were able to deliver HA into the dermis both in vitro and in vivo, whereas free HA penetrated no further than the stratum corneum. Following HANP application, HA within the skin was present in the form of free HA rather than nanoparticles. When applied in vivo, HANP significantly reduced the TEWL caused by UV irradiation. Thus, although free HA does not penetrate into the skin by passive diffusion, HA can be effectively delivered by nanoparticles. HA is then released from the nanoparticles and can contribute to barrier recovery following UV irradiation.

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.

NIR and UV-responsive degradable hyaluronic acid nanogels for CD44-targeted and remotely triggered intracellular doxorubicin delivery

Hyaluronic acid (HA) is an endogenous polysaccharide that shows intrinsic targetability to CD44+ cancer cells. Here, we developed NIR and UV-responsive degradable nanogels from hyaluronic acid-g-7-N,N-diethylamino-4-hydroxymethylcoumarin (HA-CM) for CD44 targeted and remotely controlled intracellular doxorubicin (DOX) delivery. Nanometer-sized HA-CM nanogels could readily load DOX, and both NIR and UV irradiation could significantly enhance DOX release from the nanogels, resulting from light-triggered cleavage of urethane bonds that connect CM to HA. MTT assays showed that DOX-loaded HA-CM nanogels combined with NIR irradiation induced much higher antitumor activity to MCF-7 cells (CD44+) than to U-87MG cells (CD44-) and free HA pretreated MCF-7 cells. CLSM observations confirmed that DOX-loaded HA-CM nanogels were internalized by CD44+ cells via receptor mediated endocytosis mechanism, and intracellular DOX release was triggered by NIR. These HA-CM nanogels with easy preparation, CD44 targetability and photo-controlled intracellular drug release are interesting for cancer chemotherapy.

Hyaluronic acid-serum albumin conjugate-based nanoparticles for targeted cancer therapy.

Multiple carcinomas including breast, ovarian, colon, lung and stomach cancer, overexpress the hyaluronic acid (HA) receptor, CD44. Overexpression of CD44 contributes to key cancer processes including tumor invasion, metastasis, recurrence, and chemoresistance. Herein, we devised novel targeted nanoparticles (NPs) for delivery of anticancer chemotherapeutics, comprised of self-assembling Maillard reaction-based conjugates of HA and bovine serum albumin (BSA). HA served as the hydrophilic block, and as the ligand for actively targeting cancer cells overexpressing CD44. We demonstrate that Maillard reaction-based covalent conjugates of BSA-HA self-assemble into NPs, which efficiently entrap hydrophobic cytotoxic drugs including paclitaxel and imidazoacridinones. Furthermore, BSA-HA conjugates stabilized paclitaxel and prevented its aggregation and crystallization. The diameter of the NPs was < 15 nm, thus enabling CD44 receptor-mediated endocytosis. These NPs were selectively internalized by ovarian cancer cells overexpressing CD44, but not by cognate cells lacking this HA receptor. Moreover, free HA abolished the endocytosis of drug–loaded BSA-HA conjugates. Consistently, drug-loaded NPs were markedly more cytotoxic to cancer cells overexpressing CD44 than to cells lacking CD44, due to selective internalization, which could be competitively inhibited by excess free HA. Finally, a CD44-targeted antibody which blocks receptor activity, abolished internalization of drug-loaded NPs. In conclusion, a novel cytotoxic drug-loaded nanomedicine platform has been developed, which is based on natural biocompatible biopolymers, capabale of targeting cancer cells with functional surface expression of CD44.

Nano-polyplex based on oleoyl-carboxymethy-chitosan (OCMCS) and hyaluronic acid for oral gene vaccine delivery

Here we described nano-polyplexes (NPs) made of oleoyl-carboxymethy-chitosan (OCMCS)/hyaluronic acid (HA) as novel potential carriers for oral gene vaccines delivery. Aerolysin gene (aerA) of Aeromonas hydrophila as microbial antigen was efficiently loaded to form OCMCS-HA/aerA (OHA) NPs. OHA NPs performed the optimal parameters, i.e. smallest (154.5±9.4nm), positive charged (+7.9±0.5mV) and monodispersed system with the N/P ratio of 5 and OCMCS/HA weight ratio of 4. Upon the introduction of HA, OHA NPs was beneficial for the DNA release in intestinal environments in comparison to OA NPs. The mean fluorescence intensity detected in Caco-2 cells incubated with OHA NPs was about 2.5-fold higher than that of OA NPs; however, it decreased significantly in the presence of excess free HA. The OHA NPs and OA NPs decreased the transepithelial electric resistance (TEER) of Caco-2 monolayers obviously and induced increasing the apparent permeability coefficient (Papp) of DNA by 5.45–6.09 folds compared with free DNA. Significantly higher (P<0.05) antigen-specific antibodies were detected in serum after orally immunized with OHA NPs than that immunized with OA NPs and DNA alone in carps. These results enable the OHA NPs might resolve challenges arising from gastrointestinal damage to gene antigens, and offer an approach applicable for oral vaccination.

Near infrared fluorescent nanoparticles based on hyaluronic acid: Self-assembly, optical properties, and cell interaction

Fluorescent imaging agents that can specifically highlight tumor cells could have a significant impact on image-guided tumor removal. Here, fluorescent nanoparticles (NPs) derived from hyaluronic acid (HA) are investigated. HA is a ligand for the receptor CD44, which is a common biomarker present on many primary tumor cells, cancer-initiating cells, and tumor-associated fibroblasts. In addition, a family of enzymes that degrade HA, called hyaluronidases (HYALs), are also overexpressed with increased activity in many tumors. We report the design and development of a panel of targeted imaging agents using the near-infrared (NIR) dye, Cy7.5, that was directly conjugated to hydrophobically-modified HA. Two different molecular weights of HA, 10kDa and 100kDa, and three different degrees of hydrophobic moiety conjugation (0, 10, and 30mol%) were utilized to develop a panel of NPs with variable size that ranged from 50 to 400nm hydrodynamic diameter (HD) depending HA molecular weight, extent of fluorescence quenching (25–50%), kinetics of cellular uptake, and targeting to CD44+ cells. The kinetics and energy-dependence of cellular uptake in breast and prostate cancer cell lines, MDA-MB 231 and PC-3 cells, respectively, showed increased uptake with longer incubation times (at 4 and 8h compared to 1h), as well as uptake at 37°C but not 4°C, which indicated energy-dependent endocytosis. NP uptake studies in the presence of excess free HA showed that pre-treatment of cells with excess high molecular weight (MW) free HA decreased NP uptake by up to 50%, while no such trend was observed with low MW HA. These data lay the foundation for selection of optimized HA-derived NPs for image-guided surgery. Statement of SignificanceHere, hyaluronic acid (HA), a well-studied biomacromolecule, is modified with a near infrared fluorophore and a hydrophobic moiety. The significance of this work, especially for imaging applications, is that the impact of HA molecular weight and the hydrophobic moiety conjugation degree on fluorescence and cell interaction can be predicted. With respect to existing literature, the eventual use of these HA-based NPs is image-guided surgery; thus, we focus on the dye, Cy7.5, for conjugation, which is more NIR than most existing HA literature. Furthermore, HA is a ligand for CD44, which is associated with cancer and tumor microenvironment cells. Systematic studies in this work highlight that HA can be tuned to maximize or minimize CD44 binding

Hyaluronic acid-conjugated polyamidoamine dendrimers for targeted delivery of 3,4-difluorobenzylidene curcumin to CD44 overexpressing pancreatic cancer cells

The current study was aimed to develop a targeted dendrimer formulation of 3, 4-difluorobenzylidene curcumin (CDF) and evaluate its potential in CD44 targeted therapy for pancreatic cancer. Using amine terminated fourth generation poly(amidoamine) (PAMAM) dendrimer nanocarrier and hyaluronic acid (HA) as a targeting ligand, we engineered a CD44-targeted PAMAM dendrimer (HA-PAMAM) formulation of CDF. The resulting dendrimer nanosystem (HA-PAMAM-CDF) had a particle size and surface charge of 9.3±1.5nm and −7.02±9.53mV, respectively. When CD44 receptor overexpressing MiaPaCa-2 and AsPC-1 human pancreatic cancer cells were treated with HA-PAMAM-CDF, a dose-dependent cytotoxicity was observed. Furthermore, blocking the CD44 receptors present on the MiaPaCa-2 cells using free excess soluble HA prior to treatment with HA-PAMAM-CDF nano-formulation resulted in 1.71 fold increase in the IC50 value compared to non-targeted formulation (PAMAM-CDF), confirming target specificity of HA-PAMAM-CDF. Additionally, HA-PAMAM-CDF formulation when compared to PAMAM-CDF, displayed higher cellular uptake in MiaPaCa-2 cancer cell lines as shown by fluorescence studies. In summary, the novel CD44 targeted dendrimer based nanocarriers appear to be proficient in mediating site-specific delivery of CDF via CD44 receptors, with an improved therapeutic margin and safety.

Smart Nanoparticles Based on Hyaluronic Acid for Redox-Responsive and CD44 Receptor-Mediated Targeting of Tumor.

BackgroundSince aggressive cancer cells highly express the CD44 receptor compared to normal cells, hyaluronic acid (HA) can be used for CD44 targeting molecule. Since glutathione (GSH) level is normally elevated in the intracellular compartment and in the tumor cell, the fact that disulfide bond can be cleaved by GSH is widely used for intracellular drug delivery.MethodsHA was connected with poly(dl-lactide-co-glycolide) (PLGA) using disulfide linkage, and then a diblock copolymer (HAssLG) was prepared. Doxorubicin (DOX)-loaded HAssLG nanoparticles were prepared by dialysis procedures.Results and DiscussionDOX-loaded HAssLG nanoparticles have spherical shapes with small particle size of less than 300 nm. In fluorescence measurement, DOX was dose-dependently liberated from nanoparticles by the addition of GSH. DOX release rate from HAssLG nanoparticles was increased by the addition of GSH. To confirm CD44 receptor-mediated endocytosis of nanoparticles, CD44-positive MDA-MB231 cells were employed and fluorescence intensity was strong when nanoparticles were treated to tumor cells. However, fluorescence intensity was significantly decreased through blocking of the CD44 receptor by pretreatment of cells with free HA. Fluorescence intensity of cells was increased again when GSH was added, indicating that HAssLG nanoparticles have CD44 receptor targetability and potential of redox-responsive drug delivery. For animal imaging study, CD44-positive MDA-MB231 cells and CD44-negative NIH3T3 cells were simultaneously implanted into the right flank and left flank of mice, respectively. Fluorescence intensity was significantly stronger at tumor mass of MDA-MB231 cells than solid mass of NIH3T3 cells, indicating that HAssLG nanoparticles were specifically delivered to tumor cells.ConclusionsThe results indicated that HAssLG nanoparticles have specificity against the CD44 receptor and can be used for anticancer drug targeting. We recommend HAssLG nanoparticles as a promising vehicle for cancer drug targeting.