Microalgae-Based Biohybrid Microrobot for Accelerated Diabetic Wound Healing.

A variety of wound healing platforms have been proposed to alleviate the hypoxic condition and/or to modulate the immune responses for the treatment of chronic wounds in diabetes. However, these platforms with the passive diffusion of therapeutic agents through the blood clot result in the relatively low delivery efficiency into the deep wound site. Here, a microalgae-based biohybrid microrobot for accelerated diabetic wound healing is developed. The biohybrid microrobot autonomously moves at velocity of 33.3 µm s-1 and generates oxygen for the alleviation of hypoxic condition. In addition, the microrobot efficiently bound with inflammatory chemokines of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) for modulating the immune responses. The enhanced penetration of microrobot is corroborated by measuring fibrin clots in biomimetic wound using microfluidic devices and the enhanced retention of microrobot is confirmed in the real wounded mouse skin tissue. After deposition on the chronic wound in diabetic mice without wound dressing, the wounds treated with microrobots are completely healed after 9 days with the significant decrease of inflammatory cytokines below 31% of the control level and the upregulated angiogenesis above 20 times of CD31+ cells. These results confirm the feasibility of microrobots as a next-generation platform for diabetic wound healing.

Hydrogel Microneedles Extracting Exosomes for Early Detection of Colorectal Cancer.

There are several methods for early diagnosis of tumors, such as detecting circulating tumor DNAs, detecting circulating tumor cells, or imaging with tumor-targeting contrast agents. However, these assays are time-consuming and may cause patient discomfort during the biopsy collecting process. Here, we develop a facile method for early diagnosis of tumors by extracting exosomes from interstitial fluid (ISF) using hydrogel microneedles (MNs). The hydrogel MNs expand in the skin to absorb the ISF, and the tumor exosomes contained in the ISF bind with the glypican-1 antibodies inside the hydrogel of MNs. After removing the hydrogel on the MNs, exosomes are separately purified from the ISF to analyze tumor-related biomarkers. Finally, colorectal cancer can be diagnosed by ELISA for the colorectal cancer-induced model mice. This noninvasive hydrogel MN system to obtain the exosome samples would play an important role in early cancer diagnosis.

Hyaluronate-Black Phosphorus-Upconversion Nanoparticle Complex for Non-invasive Theranosis of Skin Cancer.

Despite the wide investigation on black phosphorus (BP) for biophotonic applications, the finite depth of light penetration has limited further development of BP-based photomedicines. Here, we developed a hyaluronate-BP-upconversion nanoparticle (HA-BP-UCNP) complex for near-infrared (NIR) light-mediated multimodal theranosis of skin cancer with photoacoustic (PA) bioimaging, photodynamic therapy (PDT), and photothermal therapy (PTT). In contrast to the conventional BP-based skin cancer theranosis, the HA-BP-UCNP complex could be non-invasively delivered into the tumor tissue to induce the cancer cell apoptosis upon NIR light irradiation. The PA imaging of BP successfully visualized the non-invasive transdermal delivery of the HA-BP-UCNP complex into the mice skin. HA in the complex facilitated the transdermal delivery of BP into the tumor tissue under the skin. Upon 980 nm NIR light irradiation, the UCNP converted the light to UV-blue light to generate reactive oxygen species by sensitizing BP in the HA-BP-UCNP complex for PDT. Remarkably, 808 nm NIR irradiation with PTT triggered the apoptosis of tumor cells. Taken together, we could confirm the feasibility of the HA-BP-UCNP complex for NIR light-mediated multimodal theranosis of skin cancers.

Hyaluronic Acid Derivatives for Translational Medicines.

The recent progress in various biomaterials with unique physiological and pharmacological properties has expedited the development of translational medicines for the diagnosis, prognosis, and therapy of intractable diseases. Hyaluronic acid (HA) is one of such biomaterials that has attracted great attention due to its unique characteristics for biomedical applications. In this Perspective, we provide an overview of HA-based medicines in a variety of forms such as chemical and biological conjugates, nanoparticles, nanoparticle hybrid systems, hydrogels, and nanogels. We highlight the current-state-of-the-art strategies to design and optimize innovative HA-based medicines for their clinical translations. Finally, we discuss the challenges for technical hurdles and the future directions to expand the feasibility of HA-based translational medicines.

Multifunctional Nanodroplets Encapsulating Naphthalocyanine and Perfluorohexane for Bimodal Image-Guided Therapy.

Although nanocarriers containing perfluorocarbon (PFC) have been widely investigated as an ultrasound (US) imaging agent and a high intensity focused ultrasound (HIFU) agent, these carriers have suffered from low stability and biocompatibility limiting their further biomedical applications. Here, we developed surface cross-linked polymer nanodroplets as a HIFU therapeutic agent guided by bimodal photoacoustic (PA) and US imaging. Pluronic F127 was reacted with 4-nitrophenyl chloroformate (NPC) and mixed with naphthalocyanine (Nc) in dichloromethane, which was added into the aqueous solution of amine-functionalized six-arm-branched poly(ethylene glycol) (PEG) to form an oil-in-water emulsion for the cross-linking reaction between the terminal NPC of Pluronic F127 and the primary amine of six-arm PEG. The resulting solution was sonicated with liquid perfluorohexane (PFH) to prepare PEG cross-linked Pluronic F127 nanoparticles encapsulating Nc and PFH (Nc/PFH@PCPN). Nc/PFH@PCPN appeared to be stable without any coalescence or vaporization in the physiological condition. Upon the application of HIFU, Nc/PFH@PCPN was vaporized and showed increased US intensity for 180 min. The Nc dye in the nanodroplets enabled the stable encapsulation of PFH and the bimodal US/PA imaging. In vivo PA/US image-guided HIFU ablation therapy confirmed that the nanodroplets increased the cavitation effect, induced necrosis and apoptosis of tumor cells, and reduced tumor growth significantly for 12 days. Taken together, the multifunctional Nc/PFH@PCPN was successfully developed as a new platform for PA/US image-guided HIFU therapy.

Smart Microbubble Eluting Theranostic Stent for Noninvasive Ultrasound Imaging and Prevention of Restenosis.

A pH-responsive microbubble-eluting theranostic stent is developed for real-time ultrasound imaging of stent implanted blood vessels and dissolution of fat-rich plaques to prevent the blocking of blood vessels in rats. This smart theranostic stent can be effectively applied to facilitate noninvasive monitoring and prevent restenosis after stent implantation.

Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells.

UNLABELLED: Liver fibrosis is a common outcome of chronic liver disease that leads to liver cirrhosis and hepatocellular carcinoma. No US Food and Drug Administration-approved targeted antifibrotic therapy exists. Activated hepatic stellate cells (aHSCs) are the major cell types responsible for liver fibrosis; therefore, eradication of aHSCs, while preserving quiescent HSCs and other normal cells, is a logical strategy to stop and/or reverse liver fibrogenesis/fibrosis. However, there are no effective approaches to specifically deplete aHSCs during fibrosis without systemic toxicity. aHSCs are associated with elevated expression of death receptors and become sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. Treatment with recombinant TRAIL could be a potential strategy to ameliorate liver fibrosis; however, the therapeutic application of recombinant TRAIL is halted due to its very short half-life. To overcome this problem, we previously generated PEGylated TRAIL (TRAILPEG ) that has a much longer half-life in rodents than native-type TRAIL. In this study, we demonstrate that intravenous TRAILPEG has a markedly extended half-life over native-type TRAIL in nonhuman primates and has no toxicity in primary human hepatocytes. Intravenous injection of TRAILPEG directly induces apoptosis of aHSCs in vivo and ameliorates carbon tetrachloride-induced fibrosis/cirrhosis in rats by simultaneously down-regulating multiple key fibrotic markers that are associated with aHSCs. CONCLUSION: TRAIL-based therapies could serve as new therapeutics for liver fibrosis/cirrhosis and possibly other fibrotic diseases. (Hepatology 2016;64:209-223).

Hyaluronate-Peanut Agglutinin Conjugates for Target-Specific Bioimaging of Colon Cancer.

Colon cancer is one of the most common death-related cancers in the world. For treating colon cancer, it is crucial to detect and remove malignant lesions early. Here, we developed hyaluronate (HA)-peanut agglutinin (PNA) conjugates for the bioimaging of colon cancer. The HA-PNA conjugates were successfully synthesized by the coupling reaction between aldehyde-modified HA and the N-terminal amine group of PNA. For diagnostic imaging, rhodamine B (RhoB) was chemically conjugated onto PNA in HA-PNA conjugates. After intraluminal injection of HA-PNA-RhoB conjugates into tumor-bearing mice, small-sized colon cancers could be effectively visualized by ex vivo imaging with an in vivo imaging system (IVIS) and a two-photon microscope. With these results taken together, we could confirm the feasibility of HA-PNA-RhoB conjugates as a bioimaging agent for detecting colon cancers.

Tocilizumab-Alendronate Conjugate for Treatment of Rheumatoid Arthritis.

An autoimmune disease of rheumatoid arthritis (RA) causes severe inflammation on the synovial membrane, which results in the destruction of articular cartilage and bone. Here, Tocilizumab (TCZ)-Alendronate (ALD) conjugate is synthesized for the early intervention of RA. A humanized monoclonal antibody of TCZ shows an immunosuppressive effect, targeting interleukin-6 (IL-6) receptor in the RA pathogenesis. ALD is an anti-inflammatory bisphosphonate drug which can bind to the exposed bone surface. ALD is conjugated selectively to N-glycan on Fc region of TCZ using a chemical linker of 3-(2-pyridyldithio)propionyl hydrazide (PDPH)-poly(ethylene glycol)-N-hydroxysuccinimide (PDPH-PEG-NHS). The successful synthesis of TCZ-ALD conjugate is corroborated by 1H NMR, the purpald assay, mass spectrometry (MS), and high performance liquid chromatography (HPLC). In vitro binding affinity and cell viability tests confirmed the biological activity of TCZ-ALD conjugate. Furthermore, in vivo efficacy of TCZ-ALD conjugate is confirmed by microcomputed tomography (CT), histology, and Western blot analyses for the treatment of RA.

Noninvasive Transdermal Vaccination Using Hyaluronan Nanocarriers and Laser Adjuvant.

Vaccines are commonly administered by injection using needles. Although transdermal microneedles are less-invasive promising alternatives, needle-free topical vaccination without involving physical damage to the natural skin barrier is still sought after as it can further reduce needle-induced anxiety and simply administration. However, this long-standing goal has been elusive since the intact skin is impermeable to most macromolecules. Here, we show an efficient, non-invasive transdermal vaccination in mice by employing two key innovations: first, the use of hyaluronan (HA) as vaccine carriers and, second, non-ablative laser adjuvants. Conjugates of a model vaccine ovalbumin (OVA) and HA-HA-OVA conjugates-induced more effective maturation of dendritic cells in vitro, compared to OVA or HA alone, through synergistic HA receptor-mediated effects. Following topical administration in the back skin, HA-OVA conjugates penetrated into the epidermis and dermis in murine and porcine skins up to 30% of the total applied quantity, as revealed by intravital microscopy and quantitative fluorescence assay. Topical administration of HA-OVA conjugates significantly elevated both anti-OVA IgG antibody levels in serum and IgA antibody levels in bronchioalveolar lavage, with peak levels at 4 weeks, while OVA alone had a negligible effect. An OVA challenge at week 8 elicited strong immune-recall humoral responses. With pre-treatment of the skin using non-ablative fractional laser beams (1410 nm wavelength, 10 ms pulse duration, 0.2 mJ/pulse) as laser adjuvant, strong immunization was achieved with much reduced doses of HA-OVA (1 mg/kg OVA). Our results demonstrate the potential of the non-invasive patch-type transdermal vaccination platform.

Controlled Detachment of Chemically Glued Cells.

We demonstrate a chemically detachable cell-glue system based on linkers containing disulfide bonds as well as functional groups for metabolic glycoengineering and bioorthogonal click chemistry. Azide groups are generated on the cell surface by metabolic glycoengineering, and they are further modified into tetrazine (Tz) or trans-cyclooctene (TCO) using rationally designed cross-linkers. When the Tz-modified and TCO-modified cells are mixed together, cell gluing between these two cell groups is established by Tz-TCO click chemistry. This artificial cell-cell adhesion can be broken by the administration of glutathione (5 mM), which triggers the degradation of disulfide bonds. Both the gluing and detachment processes are rapid (<10 min) and minimally cytotoxic.

Hyaluronate-Death Receptor 5 Antibody Conjugates for Targeted Treatment of Liver Metastasis.

The liver is the most frequent site of metastasis with a 5-year survival rate of only 20-40%. In this work, hyaluronate (HA)-death receptor 5 antibody (DR5 Ab) conjugate was synthesized as a dual targeting therapeutic agent to treat liver metastasis. Dual targeting was achieved by DR5 Ab, a humanized agonistic monoclonal antibody binding to DR5 frequently overexpressed in many kinds of cancer cells, and by HA, a natural polysaccharide binding to HA receptors highly expressed in both the liver and cancer cells. Thiol end-modified HA was site-specifically conjugated to N-glycan on Fc region of oxidized DR5 Ab using a heterobifunctional linker of 3-(2-pyridyldithio)propionyl hydrazide (PDPH). The successful synthesis of HA-DR5 Ab conjugate was confirmed by (1)H NMR, purpald assay, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC). In vitro analysis of HA-DR5 Ab conjugate revealed that the conjugation of HA to DR5 Ab did not affect the binding affinity and anticancer efficacy of DR5 Ab. Remarkably, according to in vivo bioimaging study, HA-DR5 Ab conjugate appeared to be highly accumulated in the liver and dramatically effective in inhibiting the tumor growth in liver metastasis model mice.

Hyaluronate-Epidermal Growth Factor Conjugate for Skin Wound Healing and Regeneration.

Epidermal growth factor (EGF) has been recognized as an excellent wound healing agent due to its therapeutic function stimulating skin cell growth, proliferation and differentiation. However, the transdermal delivery of EGF poses a significant challenge due to its short half-life and lack of efficient formulation. Here, to improve the transdermal delivery efficiency, EGF was conjugated to hyaluronate (HA), which was formulated into a patch-type film for skin wound healing. HA-EGF conjugate was synthesized by coupling reaction between aldehyde-modified HA and N-terminal amine group of EGF to minimize the loss of biological activities. The HA-EGF conjugates exhibited similar biological activities with native EGF as confirmed by ELISA and proliferation tests using murine and human fibroblasts. For the efficient topical delivery, HA-EGF conjugates were incorporated into a matrix film of high molecular weight HA. Two-photon microscopy clearly visualized more efficient transdermal delivery of HA-EGF conjugates to both normal skin and peripheral tissues around the wound area rather than that of EGF. Optical imaging and ELISA after in vivo transdermal delivery showed that the conjugation of EGF to HA retarded its degradation and extended its residence time in the wound area. Furthermore, in vivo transdermal delivery of HA-EGF conjugate in the patch-type HA film resulted in significantly improved regeneration of skin tissues even into hypodermis.

Bioorthogonal Click Chemistry-Based Synthetic Cell Glue.

Artificial methods of cell adhesion can be effective in building functional cell complexes in vitro, but methods for in vivo use are currently lacking. Here, a chemical cell glue based on bioorthogonal click chemistry with high stability and robustness is introduced. Tetrazine (Tz) and trans-cyclooctene (TCO) conjugated to the cell surface form covalent bonds between cells within 10 min in aqueous conditions. Glued, homogeneous, or heterogeneous cell pairs remain viable and stably attached in a microfluidic flow channel at a shear stress of 20 dyn cm(-2) . Upon intravenous injection of assembled Jurkat T cells into live mice, fluorescence microscopy shows the trafficking of cell pairs in circulation and their infiltration into lung tissues. These results demonstrate the promising potential of chemically glued cell pairs for various applications ranging from delivering therapeutic cells to studying cell-cell interactions in vivo.

Bioimaging of Hyaluronate-Interferon α Conjugates Using a Non-Interfering Zwitterionic Fluorophore.

We conducted real-time bioimaging of the hyaluronate-interferon α (HA-IFNα) conjugate using a biologically inert zwitterionic fluorophore of ZW800-1 for the treatment of hepatitis C virus (HCV) infection. ZW800-1 was labeled on the IFNα molecule of the HA-IFNα conjugate to investigate its biodistribution and clearance without altering its physicochemical and targeting characteristics. Confocal microscopy clearly visualized the effective in vitro cellular uptake of the HA-IFNα conjugate to HepG2 cells. After verifying the biological activity in Daudi cells, we conducted the pharmacokinetic analysis of the HA-IFNα conjugate, which confirmed its target-specific delivery to the liver with a prolonged residence time longer than that of PEGylated IFNα. In vivo and ex vivo bioimaging of the ZW800-1-labeled HA-IFNα conjugate directly showed real-time biodistribution and clearance of the conjugate that are consistent with the biological behaviors analyzed by an enzyme-linked immunosorbent assay. Furthermore, the elevated level of OAS1 mRNA in the liver confirmed in vivo antiviral activity of HA-IFNα conjugates. With the data taken together, we could confirm the feasibility of ZW800-1 as a biologically inert fluorophore and target-specific HA-IFNα conjugate for the treatment of HCV infection.

3D tissue engineered supramolecular hydrogels for controlled chondrogenesis of human mesenchymal stem cells.

Despite a wide investigation of hydrogels as an artificial extracellular matrix, there are few scaffold systems for the facile spatiotemporal control of mesenchymal stem cells (MSCs). Here, we report 3D tissue engineered supramolecular hydrogels prepared with highly water-soluble monofunctionalized cucurbit[6]uril-hyaluronic acid (CB[6]-HA), diaminohexane conjugated HA (DAH-HA), and drug conjugated CB[6] (drug-CB[6]) for the controlled chondrogenesis of human mesenchymal stem cells (hMSCs). The mechanical property of supramolecular HA hydrogels was modulated by changing the cross-linking density for the spatial control of hMSCs. In addition, the differentiation of hMSCs was temporally controlled by changing the release profiles of transforming growth factor-ß3 (TGF-ß3) and/or dexamethasone (Dexa) from the hydrolyzable Dexa-CB[6]. The effective chondrogenic differentiation of hMSCs encapsulated in the monoCB[6]/DAH-HA hydrogel with TGF-ß3 and Dexa-CB[6] was confirmed by biochemical glycosaminoglycan content analysis, real-time quantitative PCR, histological, and immunohistochemical analyses. Taken together, we could confirm the feasibility of cytocompatible monoCB[6]/DAH-HA hydrogels as a platform scaffold with controlled drug delivery for cartilage regeneration and other various tissue engineering applications.

Biocompatible polymer-based micro/nanorobots for theranostic translational applications.

Recently, micro/nanorobots (MNRs) with self-propulsion have emerged as a promising smart platform for diagnostic, therapeutic and theranostic applications. Especially, polymer-based MNRs have attracted huge attention due to their inherent biocompatibility and versatility, making them actively explored for various medical applications. As the translation of MNRs from laboratory to clinical settings is imperative, the use of appropriate polymers for MNRs is a key strategy, which can prompt the advancement of MNRs to the next phase. In this review, we describe the multifunctional versatile polymers in MNRs, and their biodegradability, motion control, cargo loading and release, adhesion, and other characteristics. After that, we review the theranostic applications of polymer-based MNRs to bioimaging, biosensing, drug delivery, and tissue engineering. Furthermore, we address the challenges that must be overcome to facilitate the translational development of polymeric MNRs with future perspectives. This review would provide valuable insights into the state-of-the-art technologies associated with polymeric MNRs and contribute to their progression for further clinical development.

Multifunctional nanomaterials for smart wearable diabetic healthcare devices.

Wearable diabetic healthcare devices have attracted great attention for real-time continuous glucose monitoring (CGM) using biofluids such as tears, sweat, saliva, and interstitial fluid via noninvasive ways. In response to the escalating global demand for CGM, these devices enable proactive management and intervention of diabetic patients with incorporated drug delivery systems (DDSs). In this context, multifunctional nanomaterials can trigger the development of innovative sensing and management platforms to facilitate real-time selective glucose monitoring with remarkable sensitivity, on-demand drug delivery, and wireless power and data transmission. The seamless integration into wearable devices ensures patient's compliance. This comprehensive review evaluates the multifaceted roles of these materials in wearable diabetic healthcare devices, comparing their glucose sensing capabilities with conventionally available glucometers and CGM devices, and finally outlines the merits, limitations, and prospects of these devices. This review would serve as a valuable resource, elucidating the intricate functions of nanomaterials for the successful development of advanced wearable devices in diabetes management.

Strain-Programmed Adhesive Patch for Accelerated Photodynamic Wound Healing.

As an alternative to tissue adhesives, photochemical tissue bonding is investigated for advanced wound healing. However, these techniques suffer from relatively slow wound healing with bleeding and bacterial infections. Here, the versatile attributes of afterglow luminescent particles (ALPs) embedded in dopamine-modified hyaluronic acid (HA-DOPA) patches for accelerated wound healing are presented. ALPs enhance the viscoelastic properties of the patches, and the photoluminescence and afterglow luminescence of ALPs maximize singlet oxygen generation and collagen fibrillogenesis for effective healing in the infected wounds. The patches are optimized to achieve the strong and rapid adhesion in the wound sites. In addition, the swelling and shrinking properties of adhesive patches contribute to a nonlinear behavior in the wound recovery, playing an important role as a strain-programmed patch. The protective patch prevents secondary infection and skin adhesion, and the patch seamlessly detaches during wound healing, enabling efficient residue clearance. In vitro, in vivo, and ex vivo model tests confirm the biocompatibility, antibacterial effect, hemostatic capability, and collagen restructuring for the accelerated wound healing. Taken together, this research collectively demonstrates the feasibility of HA-DOPA/ALP patches as a versatile and promoting solution for advanced accelerated wound healing, particularly in scenarios involving bleeding and bacterial infections.

Cold-Responsive Hyaluronated Upconversion Nanoplatform for Transdermal Cryo-Photodynamic Cancer Therapy.

Cryotherapy leverages controlled freezing temperature interventions to engender a cascade of tumor-suppressing effects. However, its bottleneck lies in the standalone ineffectiveness. A promising strategy is using nanoparticle therapeutics to augment the efficacy of cryotherapy. Here, a cold-responsive nanoplatform composed of upconversion nanoparticles coated with silica - chlorin e6 - hyaluronic acid (UCNPs@SiO2-Ce6-HA) is designed. This nanoplatform is employed to integrate cryotherapy with photodynamic therapy (PDT) in order to improve skin cancer treatment efficacy in a synergistic manner. The cryotherapy appeared to enhance the upconversion brightness by suppressing the thermal quenching. The low-temperature treatment afforded a 2.45-fold enhancement in the luminescence of UCNPs and a 3.15-fold increase in the photodynamic efficacy of UCNPs@SiO2-Ce6-HA nanoplatforms. Ex vivo tests with porcine skins and the subsequent validation in mouse tumor tissues revealed the effective HA-mediated transdermal delivery of designed nanoplatforms to deep tumor tissues. After transdermal delivery, in vivo photodynamic therapy using the UCNPs@SiO2-Ce6-HA nanoplatforms resulted in the optimized efficacy of 79% in combination with cryotherapy. These findings underscore the Cryo-PDT as a truly promising integrated treatment paradigm and warrant further exploring the synergistic interplay between cryotherapy and PDT with bright upconversion to unlock their full potential in cancer therapy.