Hyaluronic Acid Nanogels Research Articles

EGFR and CD44 Dual-Targeted Multifunctional Hyaluronic Acid Nanogels Boost Protein Delivery to Ovarian and Breast Cancers In Vitro and In Vivo.

Protein drugs with intracellular targets like Granzyme B (GrB) have demonstrated great proliferative inhibition activity in cancer cells. Their clinical translation, however, relies on the development of safe, efficient, and selective protein-delivery vehicles. Here, we report that epidermal growth factor receptor (EGFR) and CD44 dual-targeted multifunctional hyaluronic acid nanogels (EGFR/CD44-NGs) boost protein delivery to ovarian and breast cancers in vitro and in vivo. EGFR/CD44-NGs obtained via nanoprecipitation and photoclick chemistry from hyaluronic acid derivatives with tetrazole, GE11 peptide/tetrazole, and cystamine methacrylate groups had nearly quantitative loading of therapeutic proteins like cytochrome C (CC) and GrB, a small size of ca. 165 nm, excellent stability in serum, and fast protein release under a reductive condition. Flow cytometry assays showed that EGFR/CD44-NGs exhibited over 6-fold better uptake in CD44 and EGFR-positive SKOV-3 ovarian cancer cells than CD44-NGs. In accordance, GrB-loaded EGFR/CD44-NGs (GrB-EGFR/CD44-NGs) displayed enhanced caspase activity and growth inhibition in SKOV-3 cells as compared to GrB-loaded CD44-NGs (GrB-CD44-NGs) control. Intriguingly, the therapeutic studies in SKOV-3 human ovarian carcinoma and MDA-MB-231 human breast tumor xenografted in nude mice revealed that GrB-EGFR/CD44-NGs at a low dose of 3.85 nmol GrB equiv/kg induced nearly complete growth suppression of both tumors, which was obviously more effective than GrB-CD44-NGs, without causing any adverse effects. EGFR and CD44 dual-targeted multifunctional hyaluronic acid nanogels have appeared as a safe and efficacious platform for cancer protein therapy.

Hyaluronic acid nanogels prepared via ortho ester linkages show pH-triggered behavior, enhanced penetration and antitumor efficacy in 3-D tumor spheroids

A new type of pH-triggered hyaluronic acid nanogel system (HA-NGs) was successfully developed for tumor-targeted delivery of drugs. HA-NGs were obtained by copolymerization between methacrylate HA and a new cross-linker containing ortho ester groups in an aqueous solution. The therapeutic drug (DOX) was loaded into the HA-NGs (DOX@HA-NGs) and exhibited appropriate loading of about 17.3% with a size of around 200nm. Such new pH-triggered HA-NGs are found to be highly desirable for targeted cancer therapy because it could significantly minimize the amount of premature drug release in neutral pH, and also provide a sufficient amount of drug to effectively kill the cancer cells caused by the degradation of ortho ester groups at acid pH values. Results from the cellular uptake and cytotoxicity of DOX@HA-NGs performed in two-dimensional (2D) cell culture demonstrated that DOX@HA-NGs exhibit excellent tumor homing and higher cytotoxicity. Importantly, the penetration and inhibition against three-dimensional (3D) tumor spheroids demonstrated that DOX@HA-NGs could fully penetrate into HepG2 tumor spheroids, thus leading to higher inhibition. So, such new tumor-targeting DOX@HA-NGs prepared via ortho ester linkages will exhibit excellent stability in a neutral environment, pH-triggered drug release, as well as enhanced penetration and destruction against 3D tumor spheroids, thereby making targeted cancer therapy possible.

Targetability of hyaluronic acid nanogel to cancer cells: In vitro and in vivo studies

We have, in previous work developed, characterized and evaluated the biocompatibility of an engineered hyaluronic acid nanogel. Here we assess the targetability of a hyaluronic acid nanogel towards CD44 overexpressing cells, in vitro and in vivo. Results obtained by flow cytometry and confocal fluorescence microscopy shows that nanogel is greatly internalized by non-small cancer lung cells (A549 cells), that overexpress CD44 receptors. The biodistribution and tumor targetability of the nanogel labelled with a near-infrared (NIR) probe were performed, in mice, through a non-invasive imaging system. Results revealed nanogel high targetability towards an induced subcutaneous A549 tumor. Nanogels pharmacokinetics was evaluated also in healthy animals, and Alexa Fluor 680 labelled nanogel exhibited higher accumulation in liver, kidneys and skin. Also, a comparative biodistribution study was performed, using two NIR imaging probes, Cy5.5 and Alexa Fluor 680.

Redox Responsive Hyaluronic Acid Nanogels for Treating RHAMM (CD168) Over-expressive Cancer, both Primary and Metastatic Tumors.

It remains a substantial challenge to targetedly deliver drug to both primary tumors and metastatic lesions employing a single nanoparticle delivery system. Here aiming at the receptor for hyaluronic acid mediated motility (RHAMM or CD168), a specific receptor for hyaluronic acid (HA), the bioreductive responsive HA nanogels loaded doxorubicin were prepared. The targeting effects of HA nanogels in high RHAMM-expressed cancer cells, primary and metastatic tumors were investigated. It was found that HA nanogels show a strong in vitro and in vivo RHAMM-mediated cellular uptake and drug delivery. The cellular uptake of HA nanogels in high RHAMM-expressed LNCaP and H22 cells was far more than the uptake in low RHAMM-expressed NIH3T3 cells. The IC50 value of drug-loaded HA nanogels against H22 cells was lower than that of free drug. In vivo antitumor activity examinations showed that the HA nanogels not only had significantly superior antitumor efficacy in murine H22 and human LNCaP tumor-bearing mice but also exhibited much deep tumor penetration. The drug delivery of lymph node metastasis by systemically administering HA nanogels demonstrated that the HA nanogels could sufficiently increase drug concentration in metastatic lymph node by RHAMM-HA interaction and inhibit the growth of metastatic lymph node, even completely heal malignant lymph node metastasis. Thus, RHAMM-directed drug delivery is a promising therapy route for treating both primary and metastatic tumors.

Multifunctional Click Hyaluronic Acid Nanogels for Targeted Protein Delivery and Effective Cancer Treatment in Vivo

Protein therapeutics offer a most effective treatment for many human diseases, including diabetes, cardiovascular diseases, and malignant tumors. Unlike most chemotherapeutics that often cause notorious side effects, many protein drugs possess a high specificity and reduced systemic toxicity. Notably, clinically used protein drugs are mostly limited to those that have extracellular effects. Protein drugs that have intracellular targets do represent a large family of protein biologics that have not been introduced into the clinic, because of the absence of translatable intracellular protein delivery vehicles. Here we report efficient and targeted cancer protein therapy in vivo by bioresponsive fluorescent photoclick hyaluronic acid (HA) nanogels. Two intracellular protein drugs, cytochrome c (CC) and granzyme B (GrB), are loaded into the nanogels with preserved bioactivity. CC- and GrB-loaded HA nanogels can effectively target and release proteins to CD44 positive MCF-7 and A549 cancer cells, yielding stri...

Redox-Sensitive and Intrinsically Fluorescent Photoclick Hyaluronic Acid Nanogels for Traceable and Targeted Delivery of Cytochrome c to Breast Tumor in Mice.

In spite of their high specificity and potency, few protein therapeutics are applied in clinical cancer therapy owing to a lack of safe and efficacious delivery systems. Here, we report that redox-sensitive and intrinsically fluorescent photoclick hyaluronic acid nanogels (HA-NGs) show highly efficient loading and breast tumor-targeted delivery of cytochrome c (CC). HA-NGs were obtained from hyaluronic acid-graft-oligo(ethylene glycol)-tetrazole (HA-OEG-Tet) via inverse nanoprecipitation and catalyst-free photoclick cross-linking with l-cystine dimethacrylamide (MA-Cys-MA). HA-NGs exhibited a superb CC loading content of up to 40.6 wt %, intrinsic fluorescence (λem = 510 nm), and a small size of ca. 170 nm. Notably, CC-loaded nanogels (CC-NGs) showed a fast glutathione-responsive protein release behavior. Importantly, released CC maintained its bioactivity. MTT assays revealed that CC-NGs were highly potent with a low IC50 of 3.07 μM to CD44+ MCF-7 human breast tumor cells. Confocal microscopy observed efficient and selective internalization of fluorescent HA-NGs into MCF-7 cells. Interestingly, HA-NGs exhibited also effective breast tumor penetration. The therapeutic results demonstrated that CC-NGs effectively inhibited the growth of MCF-7 breast tumor xenografts at a particularly low dose of 80 or 160 nmol CC equiv./kg. Moreover, CC-NGs did not cause any change in mice body weight, corroborating their low systemic side effects. Redox-sensitive and intrinsically fluorescent photoclick hyaluronic acid nanogels have appeared as a "smart" protein delivery nanoplatform enabling safe, efficacious, traceable, and targeted cancer protein therapy in vivo.

Biocompatibility of a Self-Assembled Crosslinkable Hyaluronic Acid Nanogel

Hyaluronic acid nanogel (HyA-AT) is a redox sensitive crosslinkable nanogel, obtained through the conjugation of a thiolated hydrophobic molecule to the hyaluronic acid chain. Engineered nanogel was studied for its biocompatibility, including immunocompatibility and hemocompatability. The nanogel did not compromise the metabolic activity or cellular membrane integrity of 3T3, microvascular endothelial cells, and RAW 264.7 cell lines, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase release assays. Also, we didn't observe any apoptotic effect on these cell lines through the Annexin V-FITC test. Furthermore, the nanogel cell internalization was analyzed using murine bone marrow derived macrophages, and the in vivo and ex vivo biodistribution of the Cy5.5 labeled nanogel was monitored using a non-invasive near-infrared fluorescence imaging system. The HyA-AT nanogel exhibits fairly a long half-live in the blood stream, thus showing potential for drug delivery applications.

Multi-functionalized hyaluronic acid nanogels crosslinked with carbon dots as dual receptor-mediated targeting tumor theranostics

Hyaluronic acid (HA)-based theranostic nanogels were designed for the tumor diagnosis and chemotherapy, by crosslinking the folate-terminated poly(ethylene glycol) modified hyaluronic acid (FA-PEG-HA) with carbon dots (CDs) for the first time. Due to the extraordinary fluorescence property of the integrated CDs, the theranostic nanogels could be used for the real-time and noninvasive location tracking to cancer cells. HA could load Doxorubicin (DOX) via electrostatic interaction with a drug-loading capacity (DLC) of 32.5%. The nanogels possessed an ideal release of DOX in the weak acid environment, while it was restrained in the neutral media, demonstrating the pH-responsive controlled release behavior. The cytotoxicity and cellular uptake results clearly illustrated that most DOX was released and accumulated in the cell nuclei and killed the cancer cells efficaciously, due to their dual receptor-mediated targeting characteristics.

Delivery of LLKKK18 loaded into self-assembling hyaluronic acid nanogel for tuberculosis treatment

Tuberculosis (TB), a disease caused by the human pathogen Mycobacterium tuberculosis, recently joined HIV/AIDS on the top rank of deadliest infectious diseases. Low patient compliance due to the expensive, long-lasting and multi-drug standard therapies often results in treatment failure and emergence of multi-drug resistant strains. In this scope, antimicrobial peptides (AMPs) arise as promising candidates for TB treatment.Here we describe the ability of the exogenous AMP LLKKK18 to efficiently kill mycobacteria. The peptide's potential was boosted by loading into self-assembling Hyaluronic Acid (HA) nanogels. These provide increased stability, reduced cytotoxicity and degradability, while potentiating peptide targeting to main sites of infection. The nanogels were effectively internalized by macrophages and the peptide presence and co-localization with mycobacteria within host cells was confirmed. This resulted in a significant reduction of the mycobacterial load in macrophages infected in vitro with the opportunistic M. avium or the pathogenic M. tuberculosis, an effect accompanied by lowered pro-inflammatory cytokine levels (IL-6 and TNF-α). Remarkably, intra-tracheal administration of peptide-loaded nanogels significantly reduced infection levels in mice infected with M. avium or M. tuberculosis, after just 5 or 10 every other day administrations. Considering the reported low probability of resistance acquisition, these findings suggest a great potential of LLKKK18-loaded nanogels for TB therapeutics.

Hyaluronic acid nanogels with enzyme-sensitive cross-linking group for drug delivery

A methacrylation strategy was employed to functionalize hyaluronic acid and prepare hyaluronic acid (HA) nanogels. Dynamic light scattering, zeta potential analyzer and electron microscopy were utilized to characterize the nanogels and their enzyme-degradability in vitro. It was found that these nanogels had a spherical morphology with the diameter of about 70nm, and negative surface potential. When doxorubicin (DOX) was loaded into the nanogels, the diameter decreased to approximately 50nm with a drug loading content of 16% and encapsulation efficiency of 62%. Cellular uptake examinations showed that HA nanogels could be preferentially internalized by two-dimensional (2D) cells and three-dimensional (3D) multicellular spheroids (MCs) which both overexpress CD44 receptor. Near-infrared fluorescence imaging, biodistribution and penetration examinations in tumor tissue indicated that the HA nanogels could efficiently accumulate and penetrate the tumor matrix. In vivo antitumor evaluation found that DOX-loaded HA nanogels exhibited a significantly superior antitumor effect.

A hyaluronic acid nanogel for photo–chemo theranostics of lung cancer with simultaneous light-responsive controlled release of doxorubicin

The combined delivery of photo- and chemo-therapeutic agents is an emerging strategy to overcome drug resistance in treating cancer, and controlled light-responsive drug release is a proven tactic to produce a continuous therapeutic effect for a prolonged duration. Here, a combination of light-responsive graphene, chemo-agent doxorubicin and pH-sensitive disulfide-bond linked hyaluronic acid form a nanogel (called a graphene-doxorubicin conjugate in a hyaluronic acid nanogel) that exerts an activity with multiple effects: thermo and chemotherapeutic, real-time noninvasive imaging, and light-glutathione-responsive controlled drug release. The nanogel is mono-dispersed with an average diameter of 120 nm as observed by using TEM and a hydrodynamic size analyzer. It has excellent photo-luminescence properties and good stability in buffer and serum solutions. Graphene itself, being photoluminescent, can be considered an optical imaging contrast agent as well as a heat source when excited by laser irradiation. Thus the nanogel shows simultaneous thermo-chemotherapeutic effects on noninvasive optical imaging. We have also found that irradiation enhances the release of doxorubicin in a controlled manner. This release synergizes therapeutic activity of the nanogel in killing tumor cells. Our findings demonstrate that the graphene-doxorubicin conjugate in the hyaluronic acid nanogel is very effective in killing the human lung cancer cell line (A549) with limited toxicity in the non-cancerous cell line (MDCK).

Thermoresponsive hyaluronic acid nanogels as hydrophobic drug carrier to macrophages

Delivery systems for macrophages are particularly attractive since these phagocytic cells play a important role in immunological and inflammatory responses, also acting as host cells for microorganisms that are involved in deadly infectious diseases, such as leishmaniasis. Hyaluronic acid (HA) is specifically recognized by macrophages that are known to express HA receptors. Therefore, in this study, we focused on HA-based nanogels as drug carriers for these cells. The drug delivery was validated in an in vivo study on mice using intravital two-photon laser scanning microscopy. HA derivatives were modified with a biocompatible oligo(ethylene glycol)-based thermoresponsive polymer to form nanogels. These HA conjugates were readily prepared by varying the molar mass of initial HA and the degree of substitution via radical-mediated thiol-ene chemistry in aqueous solution. The derivatives were shown to self-assemble into spherical gel particles with diameters ranging from 150 to 214nm above 37°C. A poorly water-soluble two-photon dye was successfully loaded into the nanogels during this self-assembly process. In vitro cellular uptake tests using a RAW 264.7 murine macrophage cell line showed successful intracellular delivery of the hydrophobic dye. After intravenous injection in mice, the nanogels circulated freely in the blood but were rapidly phagocytized within 13min by circulating macrophages and stored in the liver and spleen, as observed by two-photon microscopy. Benefit can be thus expected in using such a delivery system for the liver and spleen macrophage-associated diseases.

A novel crosslinked hyaluronic acid nanogel for drug delivery.

An amphiphilic hyaluronic acid conjugate is successfully developed based on grafting a thiolated hydrophobic molecule to the polysaccharide backbone. The engineered conjugate is capable of assembling into nanostructures once dispersed in water, with average diameter of 80.2 ± 0.4 nm (n = 5), stable up to 6 months. The thiolated HyA conjugate is reticulated by dissulfide bond with a homofunctional crosslinker-1,4-Bis(3-[2-pyridyldithio]propionamido)butane (DPDPB). The drug loading efficiency of the reticulated and non-reticulated nanogel is accessed with two hydrophobic drugs, curcumin and simvastatin. Results suggest that crosslinked nanogel exhibit higher stability upon dilution and drug loading efficiency and proves to be a redox sensitive material. The nanogels hold great potential as stealth carriers of lipophilic drugs.

Injectable in situ forming hybrid iron oxide-hyaluronic acid hydrogel for magnetic resonance imaging and drug delivery.

The development of multimodal in situ cross-linkable hyaluronic acid nanogels hybridized with iron oxide nanoparticles is reported. Utilizing a chemoselective hydrazone coupling reaction, the nanogels are converted to a macroscopic hybrid hydrogel without any additional reagent. Hydrophobic cargos remain encapsulated in the hydrophobic domains of the hybrid hydrogel without leakage. However, hydrogel degradation with hyaluronidase liberates iron oxide nanoparticles. This allows the utilization of imaging agents as tracers of the hydrogel degradation. UV-vis spectrometry and MRI studies reveal that the degradability of the hydrogels correlates with their structure. The hydrogels presented here are very promising theranostic tools for hyaluronidase-mediated delivery of hydrophobic drugs, as well as imaging of hydrogel degradation and tracking of degradation products in vivo.

Shell‐Crosslinked Hyaluronic Acid Nanogels for Live Monitoring of Hyaluronidase Activity In Vivo

A hyaluronidase (HAdase) has been noticed as a potential drug target as well as prognostic marker because of its close associations with tumor invasion, metastasis, and angiogenesis. Accordingly, precise monitoring of HAdase activity in vivo seems to be crucial not only for the evaluation of HAdase activity but also for non-invasive molecular imaging. In our study, we propose a new organic, near-infrared fluorescence imaging probe, indocyanine green (ICG)-based stimuli-responsive fluorescence probe for selective imaging of HAdases with appreciable signal-to-noise (S/N) ratios in serum and in vivo. Shell-crosslinked hyaluronic acid (HA) nanogels (sc-nanogels) are generated via a reducible covalent linkage which incorporate ICG derivatives. The ICG-embeded HA nanogels via shell-crosslinking have preferable properties for ideal selective imaging and detection of HAdase activity in vivo. The sc-nanogels exhibit prominent chemical stability against external light, greatly control background signals in serum, and small size compared to use of self-assembled ICG-based carriers. Collapsed ICG in the hydrogel core is selectively disentangled by HAdase treatment for selective near-infrared imaging without unwanted background signal. The newly designed sc-nanogels may have great potential to serve as probes for improved selective imaging of HAdase-associated diseases in clinics as well as HAdase-activity screening in vivo.

Thiol-ene clickable hyaluronans: From macro-to nanogels

The fabrication of hyaluronic acid (HA) nanogels using a thiol-ene reaction has been demonstrated. HA was modified with pentenoate groups and then cross-linked with poly(ethylene glycol)-bis(thiol) by exposure to UV light. The cross-linking density and thereby the rigidity of the obtained gels were precisely controlled by the degree of substitution of pentenoate-modified HA. Their swelling properties also depended on cross-linking density. To produce hydrogels at the nanoscale, hyaluronic acid precursors were solely confined inside liposomes before cross-linking and purified after cross-linking. The size of the resulting nanogels followed their swelling properties and was also affected by their cross-linking density. Such bionanogels with tunable mechanical and swelling properties have potential in drug delivery.

Hyaluronic acid and alginate covalent nanogels by template cross-linking in polyion complex micelle nanoreactors

Hyaluronic acid (HA) and alginate (AL) covalent nanogels cross-linked with l-lysine ethyl ester were prepared by template chemical cross-linking of the polysaccharide in polyion complex micelle (PIC) nanoreactors. By using this method we were able to prepare HA and AL nanogels without organic solvents. PICs were prepared by using poly(ethylene oxide)-block-poly[(3-acrylamidopropyl)-trimethylammonium chloride] (PEO-b-PAMPTMA) or poly[(N-isopropylacrylamide)-block-PAMPTMA] (PNIPAAM-b-PAMPTMA). Only PNIPAAM-b-PAMPTMA block copolymers allowed to prepare PIC with small and controlled size. Short polysaccharide chains (Xn=50 and 63 for AL and HA, respectively, where Xn is the number of monosaccharidic units present in the polysaccharide) where used to optimize PIC formation. The remarkable difference in charge density and rigidity of HA and AL did not have a significant influence on the formation of PICs. PICs with small size (diameter of about 50–80nm) and low polydispersity were obtained up to 5mg/mL of polymer. After cross-linking with l-lysine ethyl ester, the nanoreactors were dissociated by adding NaCl. The nanogels were easily purified and isolated by dialysis. The dissociation of the nanoreactors and the formation of the nanogels were confirmed by 1H NMR, DLS, TEM and ζ-potential measurements. The size of the smallest nanogels in solution in the swollen state was 50–70nm in presence of salt and 80–100nm in water.

Injectable Hydrogel for Sustained Protein Release by Salt‐Induced Association of Hyaluronic Acid Nanogel

A hyaluronic acid-based anionic nanogel formed by self-assembly of cholesteryl-group-bearing HA is designed for protein delivery. The HA nanogel spontaneously binds various types of proteins without denaturation, such as recombinant human growth hormone, erythropoietin, exendin-4, and lysozyme. The HA nanogel shows unique colloidal properties, in particular that an injectable hydrogel is formed by salt-induced association of the HA nanogel. A pharmacokinetic study in rats shows that an in situ gel formulation, prepared by simply mixing rhGH and HA nanogel in phosphate buffer, maintains plasma rhGH levels within a narrow range over one week. Therefore, HA nanogels offer a simple method for easy formulation of therapeutic proteins and are effective for sustained protein release systems.

Indocyanine green encapsulated nanogels for hyaluronidase activatable and selective near infrared imaging of tumors and lymph nodes

Indocyanine green (ICG) encapsulated hyaluronic acid (HA) nanogels were first studied for highly selective detection of specific cancers and lymph nodes via hyaluronidase sensitive switch-on of near infrared fluorescence as a long-lasting and stimuli-responsive imaging probe.

Preparation of hyaluronic acid nanoparticles via hydrophobic association assisted chemical cross-linking—an orthogonal modular approach

The objective of this study was to develop an efficient and stable drug delivery nanocarrier based on a dually functionalized hyaluronic acid (HA) derivative which could be used as a long circulating drug delivery vehicle. Self-assembled HA nanoparticles (HA NPs) were prepared by attaching pyrene to the HA backbone and the obtained physical NPs were stabilized by chemical cross-linking of the HA chains to form hydrophobic core–hydrophilic shell NPs. Orthogonal chemoselective reactions were applied for conversion of HA into its amphiphilic derivative and subsequent cross-linking of the formed micellar-type associates. Chemical stabilization of the physical HA associates afforded therefore very stable nanoparticles that could easily be re-suspended in aqueous media after freeze-drying. In contrast, freeze-drying of the uncross-linked physically associated particles resulted in a non-soluble material. Doxorubicin (DOX), a typical anticancer drug, was entrapped into HA NPs via ionic and/or hydrophobic interactions and used for in vitrodrug release. Higher loading efficiency and the slower release profile of DOX from HA NPs were obtained with the hydrophobically encapsulated drug. We have shown that free HA NPs were readily taken up by NIH 3T3 cells without causing any toxicity to the cells, while the DOX-loaded HA NPs resulted in increased cell death comparable to the free drug. This study clearly showed the applicability of orthogonal chemoselective modifications for the synthesis of stable HA nanogel particles as a potential cancer-targeted drug delivery system.