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Abstract Adjuvants, materials added to vaccines to enhance the resulting immune response, are important components of vaccination that are many times overlooked. While vaccines always include an antigen to tell the body what to vaccinate to, of equal importance the adjuvant provides the how, a significant factor in producing a complete response. The adjuvant space has been slow to develop with the first use of an adjuvant in a licensed vaccine occurring in the 1930s, and remaining the only adjuvant in licensed vaccines for the next 80 years. However, with vaccination at the forefront of protection against new and complex pathogens, it is important to consider all components when designing an effective vaccine. Here we summarize the adjuvant space in licensed vaccines as well as the novel adjuvant space in clinical trials with a specific focus on the materials utilized and their resulting impact on the immune response. We discuss five major categories of adjuvant materials: aluminum salts, nanoparticles, viral vectors, TLR agonists, and emulsions. For each category, we delve into the current clinical trials space, the impact of these materials on vaccination, as well as some of the ways in which they could be improved. Adjuvants present an exciting opportunity to improve vaccine responses and stability, this review will help inform about the current progress of this space. Translational impact statement In the aftermath of the COVID‐19 pandemic, vaccines for infectious diseases have come into the spotlight. While antigens have always been an important focus of vaccine design, the adjuvant is a significant tool for enhancing the immune response to the vaccine that has been largely underdeveloped. This article provides a broad review of the history of adjuvants and, the current vaccine adjuvant space, and the progress seen in adjuvants in clinical trials. There is specific emphasis on the material landscape for adjuvants and their resulting mechanism of action. Looking ahead, while the novel vaccine adjuvant space features exciting new technologies and materials, there is still a need for more to meet the protective needs of new and complex pathogens.

Abstract Vaccines are an important tool in the rapidly evolving repertoire of immunotherapies in oncology. Although cancer vaccines have been investigated for over 30 years, very few have achieved meaningful clinical success. However, recent advances in areas such antigen identification, formulation development and manufacturing, combination therapy regimens, and indication and patient selection hold promise to reinvigorate the field. Here, we provide a timely update on the clinical status of cancer vaccines. We identify and critically analyze 360 active trials of cancer vaccines according to delivery vehicle, antigen type, indication, and other metrics, as well as highlight eight globally approved products. Finally, we discuss current limitations and future applications for clinical translation of cancer vaccines.

Abstract The covalent attachment of polyethylene glycol (PEG) to therapeutic agents, termed PEGylation, is a well‐established and clinically proven drug delivery approach to improve the pharmacokinetics and pharmacodynamics of drugs. Specifically, PEGylation can improve the parent drug's solubility, extend its circulation time, and reduce its immunogenicity, with minimal undesirable properties. PEGylation technology has been applied to various therapeutic modalities including small molecules, aptamers, peptides, and proteins, leading to over 30 PEGylated drugs currently used in the clinic and many investigational PEGylated agents under clinical trials. Here, we summarize the diverse types of PEGylation strategies, the key advantages of PEGylated therapeutics over their parent drugs, and the broad applications and impacts of PEGylation in clinical settings. A particular focus has been given to the size, topology, and functionalities of PEG molecules utilized in clinically used PEGylated drugs, as well as those under clinical trials. An additional section has been dedicated to analyzing some representative PEGylated drugs that were discontinued at different stages of clinical studies. Finally, we critically discuss the current challenges faced in the development and clinical translation of PEGylated agents.

Abstract Digital therapeutics are emerging as a new form of therapeutic interventions. Unlike conventional therapeutics, digital therapeutics deliver interventions directly to patients using an evidence‐based, clinically evaluated software to treat, manage, or prevent diseases. Digital therapeutics manifest in diverse forms such as web‐based applications, mobile applications on smart devices, virtual reality, and video games. As its own product category for FDA approval, digital therapeutics can function as stand‐alone treatments or in combination with conventional therapeutics to improve adherence and/or efficacy. Here, we review the clinical landscape of digital therapeutics. We summarize FDA‐approved products and their clinical use, overview >300 ongoing clinical trials, and discuss challenges for their clinical translation and strategies to overcome the same.

Abstract Red blood cell (RBC) hitchhiking has great potential in enhancing drug therapy, by improving targeting and reducing rapid clearance of nanoparticles (NPs). However, to improve the potential for clinical translation of RBC hitchhiking, a more thorough understanding of the RBC-NP interface is needed. Here, we evaluate the effects of NP surface parameters on the success and biocompatibility of NP adsorption to extracted RBCs from various species. Major differences in RBC characteristics between rabbit, mouse and human were proven to significantly impact NP adsorption outcomes. Additionally, the effects of NP design parameters, including NP hydrophobicity, zeta potential, surfactant concentration and drug encapsulation, on RBC hitchhiking are investigated. Our studies demonstrate the importance of electrostatic interactions in balancing NP adsorption success and biocompatibility. We further investigated the effect of varying the anti-coagulant used for blood storage. The results presented here offer new insights into the parameters that impact NP adsorption on RBCs that will assist researchers in experimental design choices for using RBC hitchhiking as drug delivery strategy.

Abstract Uncontrolled bleeding is a life‐threatening emergency that requires immediate intervention. Currently available on‐site bleeding interventions largely rely on the use of tourniquets, pressure dressing, and other topical hemostatic agents, which can only treat bleeding injuries that are known, accessible, and potentially compressible. Synthetic hemostats that are stable at room temperature, easy to carry, field‐usable, and able to stop internal bleeding at multiple or unknown sources, are still lacking. We recently developed a hemostatic agent via polymer peptide interfusion (HAPPI), which can selectively bind to activated platelets and injury sites after intravascular administration. Here we report that HAPPI is highly effective in treating multiple lethal traumatic bleeding conditions in normal as well as hemophilia models via either systemic administration or topical application. In a rat liver traumatic model, intravenous injection of HAPPI resulted in a significant decrease in blood loss and a four‐fold reduction in mortality rate within 2 h after injury. When applied topically on liver punch biopsy wounds in heparinized rats, HAPPI achieved a 73% of reduction in blood loss and a five‐fold increase in survival rate. HAPPI also exhibited hemostatic efficacy in hemophilia A mice by reducing blood loss. Further, HAPPI worked synergistically with rFVIIa to induce immediate hemostasis and 95% reduction in total blood loss compared to the saline‐treated group in hemophelia mice models. These results demonstrate that HAPPI is a promising field‐usable hemostatic agent for a broad range of different hemorrhagic conditions.

Abstract Periodontitis induced by chronic subgingival infection is a ubiquitous disease that causes systemic inflammatory consequences and poses a negative impact on quality of life. The disease is treated and potentially prevented by patient's self‐care aimed at eliminating the oral pathogens from the region. Currently available products for interdental self‐care, including dental floss and interdental brush, have limited ability to prevent the disease. Here, we report a coated dental floss thread, termed “nanofloss,” which uses polyphenol‐based nanocoating to functionalize the floss thread with therapeutic agents. Multiple therapeutics can be integrated into the nanofloss including antibacterial small molecules and proteins. Flossing with nanofloss‐delivered therapeutic agents to the challenging subgingival region with long‐term retention even against the flushing action of the oral fluid in vivo. Our in vitro and in vivo studies demonstrate that chlorhexidine gluconate‐loaded nanofloss effectively treats the subgingival infection by Porphyromonas gingivalis. Collectively, the nanofloss offers a promising and easily usable tool for targeted self‐care of subgingival infection against periodontitis.

Abstract Proteins are among the most common therapeutics for the treatment of diabetes, autoimmune diseases, cancer, and metabolic diseases, among others. Despite their common use, current protein therapies, most of which are injectables, have several limitations. Large proteins such as monoclonal antibodies (mAbs) suffer from poor absorption after subcutaneous injections, thus forcing their administration by intravenous injections. Even small proteins such as insulin suffer from slow pharmacokinetics which poses limitations in effective management of diabetes. Here, a deep eutectic‐based delivery strategy is used to offer a generalized approach for improving protein absorption after subcutaneous injections. The lead formulation enhances absorption of mAbs after subcutaneous injections by ≈200%. The same composition also improves systemic absorption of subcutaneously injected insulin faster than Humalog, the current gold‐standard of rapid acting insulin. Mechanistic studies reveal that the beneficial effect of deep eutectics on subcutaneous absorption is mediated by their ability to reduce the interactions of proteins with the subcutaneous matrix, especially collagen. Studies also confirm that these deep eutectics are safe for subcutaneous injections. Deep eutectic‐based formulations described here open new possibilities for subcutaneous injections of therapeutic proteins.

Abstract Ribonucleic acid (RNA) therapeutics are being actively researched as a therapeutic modality in preclinical and clinical studies. They have become one of the most ubiquitously known and discussed therapeutics in recent years in part due to the ongoing coronavirus pandemic. Since the first approval in 1998, research on RNA therapeutics has progressed to discovering new therapeutic targets and delivery strategies to enhance their safety and efficacy. Here, we provide an overview of the current clinically relevant RNA therapeutics, mechanistic basis of their function, and strategies to improve their clinical use. We discuss the 17 approved RNA therapeutics and perform an in‐depth analysis of the 222 ongoing clinical trials, with an emphasis on their respective mechanisms and disease areas. We also provide perspectives on the challenges for clinical translation of RNA therapeutics and suggest potential strategies to address these challenges.

Abstract Alzheimer's disease (AD) and Parkinson's disease (PD) are the most prevalent neurodegenerative diseases, affecting millions and costing billions each year in the United States alone. Despite tremendous progress in developing therapeutics that manage the symptoms of these two diseases, the scientific community has yet to develop a treatment that effectively slows down, inhibits, or cures neurodegeneration. To gain a better understanding of the current therapeutic frontier for the treatment of AD and PD, we provide a review on past and present therapeutic strategies for these two major neurodegenerative disorders in the clinical trial process. We briefly recap currently US Food and Drug Administration‐approved therapies, and then explore trends in clinical trials across the variables of therapy mechanism of disease intervention, administration route, use of delivery vehicle, and outcome measures, across the clinical phases over time for “Drug” and “Biologic” therapeutics. We then present the success rate of past clinical trials and analyze the intersections in therapeutic approaches for AD and PD, revealing the shift in clinical trials away from therapies targeting neurotransmitter systems that provide symptomatic relief, and towards anti‐aggregation, anti‐inflammatory, anti‐oxidant, and regeneration strategies that aim to inhibit the root causes of disease progression. We also highlight the evolving distribution of the types of “Biologic” therapies investigated, and the slowly increasing yet still severe under‐utilization of delivery vehicles for AD and PD therapeutics. We then briefly discuss novel preclinical strategies for treating AD and PD. Overall, this review aims to provide a succinct overview of the clinical landscape of AD and PD therapies to better understand the field's therapeutic strategy in the past and the field's evolution in approach to the present, to better inform how to effectively treat AD and PD in the future.

Abstract The COVID‐19 public health crisis has spotlighted the need to improve global hygiene and sanitization. In addition to causing staggering rates of transmission and fatality, COVID‐19 has severely impacted the quality of life and mental health of global citizens. The World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC) encourage hand hygiene as the first defense against the spread of infection, yet frequent handwashing is often impractical. Widely used ethanol‐based hand sanitizers provide immediate protection against pathogens on the skin, albeit short‐lived, due to their rapid evaporation. Herein, a novel, long‐lasting skin protectant formulated with biocompatible ionic liquid/deep eutectic solvents prepared using generally recognized as safe materials – choline and geranic acid (CG‐101, 5% w/w) – is described. In vitro studies demonstrated that CG‐101 inactivates bacteria and the human coronavirus, hCoV229E, for 4 h after application. Two human clinical studies demonstrate that CG‐101 does not cause skin irritation or sensitization, and a single application of CG‐101 gel imparts skin protection against microbes for significantly longer than conventional 70% ethanol‐based hand sanitizers. These data are the first to indicate that CG‐101 may be a better alternative to alcohol‐based hand sanitizers for long‐term skin protection against infectious diseases.

Abstract Adeno‐associated virus (AAV)‐mediated gene therapy is a promising therapeutic modality for curing many diseases including monogenic diseases. However, limited tissue‐targeting and restricted re‐administration due to the vector immunogenicity largely restrict its therapeutic potential. Here, using a red blood cell (RBC) as the carrier vehicle for AAV is demonstrated to improve its tissue‐targeted transduction and enable its re‐administration. Anchoring AAV to the RBC surface minimally affected its infectability toward endothelial cells. Meanwhile, AAV anchored onto RBCs is predominantly delivered to and shows efficient transduction in the lungs by virtue of the biophysical features of RBCs. RBC‐anchored AAVs lead to a four‐ to five‐fold enhancement in target gene expression in the lungsas compared to free AAVs following a single‐ or dual‐dosing regimen. While RBC anchoring does not prevent the induction of adaptive immune responses against AAV, it results in successful transgene expression upon re‐administration following prior AAV exposure. The ability to re‐administer is partially attributed to the delayed and reduced AAV neutralization by neutralizing antibodies, resulting from the combination of limited exposure of physically confined AAVs and the short time required to reach the lungs. This study's findings suggest that the RBC‐mediated approach is a promising strategy for repetitive, targeted AAV gene therapy.

Abstract Reduction of elastin in the skin causes various skin diseases as well as wrinkles and sagging with aging. Sialidase is a hydrolase that cleaves a sialic acid residue from sialoglycoconjugate. Cleavage of sialic acid from microfibrils by the sialidase isozyme Neu1 facilitates elastic fiber assembly. In the present study, we showed that a lower layer of the dermis and muscle showed relatively intense sialidase activity. The sialidase activity in the skin decreased with aging. Choline and geranate (CAGE), one of the ionic liquids, can deliver the sialidase subcutaneously while maintaining the enzymatic activity. The elastin level in the dermis was increased by applying sialidase from Arthrobacter ureafaciens (AUSA) with CAGE on the skin for 5 days in rats and senescence-accelerated mice prone 1 and 8. Sialidase activity in the dermis was considered to be mainly due to Neu2 based on the expression level of sialidase isozyme mRNA. Transdermal administration of Neu2 with CAGE also increased the level of elastin in the dermis. Therefore, not only Neu1 but also Neu2 would be involved in elastic fiber assembly. Transdermal administration of sialidase is expected to be useful for improvement of wrinkles and skin disorders due to the loss of elastic fibers.

We report the topical administration of sponge Haliclona sp. Spicules (SHS) combined with cationic flexible liposomes (CFL) to increase the delivery of small interfering RNA (siRNA) into viable skin cells in vitro and in vivo. SHS can be applied topically as novel microneedles to overcome skin barrier by creating plenty of new microchannels in stratum corneum. Subsequently, well-designed CFL can be also utilized topically as nanocarriers to overcome skin cells membrane by delivering siRNA to skin deep layers through these microchannels and thereby facilitating their cell internalization. The topical application of SHS in combination with CFL (0.05% of lipids, w/v), referred to as CFL(0.05%), enhanced siRNA skin penetration in vitro by 72.95 ± 2.97-fold compared to control group (p < 0.001). Further, the topical application of SHS in combination with CFL(0.05%) on female BALB/c mice skin resulted in 29.21% ± 1.41% of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) knockdown at all application area in vivo, which was not significantly different from the GAPDH protein knockdown rate in the subcutaneous injection center. However, the high knockdown rate only appears in the vicinity (

Abstract Gene therapies are currently one of the most investigated therapeutic modalities in both the preclinical and clinical settings and have shown promise in treating a diverse spectrum of diseases. Gene therapies aim at introducing a gene material in target cells and represent a promising approach to cure diseases that were thought to be incurable by conventional modalities. In many cases, a gene therapy requires a vector to deliver gene therapeutics into target cells; viral vectors are among the most widely studied vectors owing to their distinguished advantages such as outstanding transduction efficiency. With decades of development, viral vector‐based gene therapies have achieved promising clinical outcomes with many products approved for treating a range of diseases including cancer, infectious diseases and monogenic diseases. In addition, a number of active clinical trials are underway to further expand their therapeutic potential. In this review, we highlight the diversity of viral vectors, review approved products, and discuss the current clinical landscape of in vivo viral vector‐based gene therapies. We have reviewed 13 approved products and their clinical applications. We have also analyzed more than 200 active trials based on various viral vectors and discussed their respective therapeutic applications. Moreover, we provide a critical analysis of the major translational challenges for in vivo viral vector‐based gene therapies and discuss possible strategies to address the same.

Abstract Antiproliferative chemotherapeutic agents offer a potential effective treatment for inflammatory arthritis. However, their clinical application is limited by high systemic toxicity, low joint bioavailability as well as formulation challenges. Here, we report an intra‐articular drug delivery system combining hyaluronic acid hydrogels and drug nanocrystals to achieve localized and sustained delivery of an antiproliferative chemotherapeutic agent camptothecin for long‐term treatment of inflammatory arthritis. We synthesized a biocompatible, in situ‐forming injectable hyaluronic acid hydrogel using a naturally occurring click chemistry: cyanobenzothiazole/cysteine reaction, which is the last step reaction in synthesizing D‐luciferin in fireflies. This hydrogel was used to encapsulate camptothecin nanocrystals (size of 160–560 nm) which released free camptothecin in a sustained manner for 4 weeks. In vivo studies confirmed that the hydrogel remained in the joint over 4 weeks. By using the collagen‐induced arthritis rat model, we demonstrate that the hydrogel‐camptothecin formulation could alleviate arthritis severity as indicated by the joint size and interleukin‐1β level in the harvested joints, as well as from histological and microcomputed tomography evaluation of joints. The hydrogel‐nanocrystal formulation strategy described here offers a potential solution for intra‐articular therapy for inflammatory arthritis.

Abstract Nobiletin (NOB), a flavonoid, has extremely low water solubility and low oral bioavailability; however, despite these problems, various physiological effects have been investigated in vitro. In the present study, we investigated the transdermal delivery of NOB using choline and geranic acid (CAGE), which is a biocompatible material that has been reported to be a promising transdermal delivery approach. The feasibility was evaluated by a set of in vitro and in vivo tests. A solubility evaluation demonstrated that CAGE induced excellent solubility of NOB induced by multipoint hydrogen bonding between NOB and CAGE. In vitro transdermal tests using a Franz diffusion cell showed that CAGE was effective in enhancing transdermal absorption of NOB, compared to other penetration enhancers. Subsequent in vivo tests demonstrated that CAGE significantly improved area under the concentration-time curve of NOB in vivo and NOB/CAGE sample showed 20-times higher bioavailability than oral administration of NOB crystal. Furthermore, NOB/CAGE sample also showed significant drops of the blood glucose level in rats derived from hypoglycemic activity of NOB. Thus, transdermal administration of NOB using CAGE was shown to be feasible, which indicates that the use of CAGE may be adapted for other flavonoids that also show both low water solubility and low permeability.

Abstract The use of ionic liquids and deep eutectic solvents in biomedical applications has grown dramatically in recent years due to their unique properties and their inherent tunability. This review will introduce ionic liquids and deep eutectics and discuss their biomedical applications, namely solubilization of drugs, creation of active pharmaceutical ingredients, delivery of pharmaceuticals through biological barriers, stabilization of proteins and other nucleic acids, antibacterial agents, and development of new biosensors. Current challenges and future outlooks are discussed, including biocompatibility, the potential impact of the presence of impurities, and the importance of understanding the microscopic interactions in ionic liquids in order to design task‐specific solvents.

Abstract Nanoparticles are used in the clinic to treat cancer, resolve mineral deficiencies, image tissues, and facilitate vaccination. As a modular technology, nanoparticles combine diagnostic agents or therapeutics (e.g., elements, small molecules, biologics), synthetic materials (e.g., polymers), and biological molecules (e.g., antibodies, peptides, lipids). Leveraging these parameters, nanoparticles can be designed and tuned to navigate biological microenvironments, negotiate biological barriers, and deliver therapeutics or diagnostic agents to specific cells and tissues in the body. Recently, with the Emergency Use Authorization of the COVID‐19 lipid nanoparticle vaccines, the advantages and potential of nanoparticles as a delivery vehicle have been displayed at the forefront of biotechnology. Here, we provide a 5‐year status update on our original “Nanoparticles in the Clinic” review (also a 2‐year update on our second “Nanoparticles in the Clinic” review) by discussing recent nanoparticle delivery system approvals, highlighting new clinical trials, and providing an update on the previously highlighted clinical trials.

Abstract A growing variety of biological macromolecules are in development for use as active ingredients in topical therapies and vaccines. Dermal delivery of biomacromolecules offers several advantages compared to other delivery methods, including improved targetability, reduced systemic toxicity, and decreased degradation of drugs. However, this route of delivery is hampered by the barrier function of the skin. Recently, a large body of research has been directed toward improving the delivery of macromolecules to the skin, ranging from nucleic acids (NAs) to antigens, using noninvasive means. In this review, we discuss the latest formulation‐based efforts to deliver antigens and NAs for vaccination and treatment of skin diseases. We provide a perspective of their advantages, limitations, and potential for clinical translation. The delivery platforms discussed in this review may provide formulation scientists and clinicians with a better vision of the alternatives for dermal delivery of biomacromolecules, which may facilitate the development of new patient‐friendly prophylactic and therapeutic medicines.

Abstract Cell therapies have emerged as a promising therapeutic modality with the potential to treat and even cure a diverse array of diseases. Cell therapies offer unique clinical and therapeutic advantages over conventional small molecules and the growing number of biologics. Particularly, living cells can simultaneously and dynamically perform complex biological functions in ways that conventional drugs cannot; cell therapies have expanded the spectrum of available therapeutic options to include key cellular functions and processes. As such, cell therapies are currently one of the most investigated therapeutic modalities in both preclinical and clinical settings, with many products having been approved and many more under active clinical investigation. Here, we highlight the diversity and key advantages of cell therapies and discuss their current clinical advances. In particular, we review 28 globally approved cell therapy products and their clinical use. We also analyze >1700 current active clinical trials of cell therapies, with an emphasis on discussing their therapeutic applications. Finally, we critically discuss the major biological, manufacturing, and regulatory challenges associated with the clinical translation of cell therapies.

Abstract Choline geranate deep eutectic solvent/ionic liquid (CAGE) has shown several desirable therapeutic properties including antimicrobial activity and ability to deliver drugs transdermally in research laboratories. Here, we describe the first report of clinical translation of CAGE from the lab into the clinic for the treatment of rosacea, a common chronic inflammatory skin disorder that affects the face. We describe the seven steps of clinical translation including (a) scale‐up, (b) characterization, (c) stability analysis, (d) mechanism of action, (e) dose determination, (f) GLP toxicity study, and (g) human clinical study. We describe the challenges and outcomes in these steps, especially those that uniquely arise from the deep eutectic nature of CAGE. Our translational efforts led to a 12‐week open‐label phase 1b cosmetic study with CAGE1:2 gel (CGB400) in mild–moderate facial rosacea in 26 patients where CGB400 exhibited a marked reduction in the number of inflammatory lesions. These results demonstrate the therapeutic potential of CGB400 for treating rosacea as well as it provides insights into the translational journey of deep eutectic solvents, in particular CAGE, for dermatological applications.

Abstract Cancer therapy is increasingly shifting toward targeting the tumor immune microenvironment and influencing populations of tumor infiltrating lymphocytes. Breast cancer presents a unique challenge as tumors of the triple‐negative breast cancer subtype employ a multitude of immunosilencing mechanisms that promote immune evasion and rapid growth. Treatment of breast cancer with chemotherapeutics has been shown to induce underlying immunostimulatory responses that can be further amplified with the addition of immune‐modulating agents. Here, we investigate the effects of combining doxorubicin (DOX) and gemcitabine (GEM), two commonly used chemotherapeutics, with monophosphoryl lipid A (MPLA), a clinically used TLR4 adjuvant derived from liposaccharides. MPLA was incorporated into the lipid bilayer of liposomes loaded with a 1:1 molar ratio of DOX and GEM to create an intravenously administered treatment. In vivo studies indicated excellent efficacy of both GEM‐DOX liposomes and GEM‐DOX‐MPLA liposomes against 4T1 tumors. In vitro and in vivo results showed increased dendritic cell expression of CD86 in the presence of liposomes containing chemotherapeutics and MPLA. Despite this, a tumor rechallenge study indicated little effect on tumor growth upon rechallenge, indicating the lack of a long‐term immune response. GEM/DOX/MPLA‐L displayed remarkable control of the primary tumor growth and can be further explored for the treatment of triple‐negative breast cancer with other forms of immunotherapy.

Abstract Colorectal cancer, common in both men and women, occurs when tumors form in the linings of the colon. Common treatments of colorectal cancer include surgery, chemotherapy, and radiation therapy; however, many colorectal cancer treatments often damage healthy tissues and cells, inducing severe side effects. Conventional chemotherapeutic agents such as doxorubicin (Dox) can be potentially used for the treatment of colorectal cancer; however, they suffer from limited targeting and lack of selectivity. Here, we report that doxorubicin complexed to hyaluronic acid (HA) (HA‐Dox) exhibits an unusual behavior of high accumulation in the intestines for at least 24 hr when injected intravenously. Intravenous administrations of HA‐Dox effectively preserved the mucosal epithelial intestinal integrity in a chemical induced colon cancer model in mice. Moreover, treatment with HA‐Dox decreased the expression of intestinal apoptotic and inflammatory markers. The results suggest that HA‐Dox could effectively inhibit the development of colorectal cancer in a safe manner, which potentially be used a promising therapeutic option.

Abstract Injectable hydrogels are one of the most widely investigated and versatile technologies for drug delivery and tissue engineering applications. Hydrogels’ versatility arises from their tunable structure, which has been enabled by considerable advances in fields such as materials engineering, polymer science, and chemistry. Advances in these fields continue to lead to invention of new polymers, new approaches to crosslink polymers, new strategies to fabricate hydrogels, and new applications arising from hydrogels for improving healthcare. Various hydrogel technologies have received regulatory approval for healthcare applications ranging from cancer treatment to aesthetic corrections to spinal fusion. Beyond these applications, hydrogels are being studied in clinical settings for tissue regeneration, incontinence, and other applications. Here, we analyze the current clinical landscape of injectable hydrogel technologies, including hydrogels that have been clinically approved or are currently being investigated in clinical settings. We summarize our analysis to highlight key clinical areas that hydrogels have found sustained success in and further discuss challenges that may limit their future clinical translation.

Abstract Nanoparticle‐based therapeutic formulations are being increasingly explored for the treatment of various ailments. Despite numerous advances, the success of nanoparticle‐based technologies in treating brain diseases has been limited. Translational hurdles of nanoparticle therapies are attributed primarily to their limited ability to cross the blood–brain barrier (BBB), which is one of the body's most exclusive barriers. Several efforts have been focused on developing affinity‐based agents and using them to increase nanoparticle accumulation at the brain endothelium. Very little is known about the role of fundamental physical parameters of nanoparticles such as size, shape, and flexibility in determining their interactions with and penetration across the BBB. Using a three‐dimensional human BBB microfluidic model (μHuB), we investigate the impact of these physical parameters on nanoparticle penetration across the BBB. To gain insights into the dependence of transport on nanoparticle properties, two separate parameters were measured: the number of nanoparticles that fully cross the BBB and the number that remain associated with the endothelium. Association of nanoparticles with the brain endothelium was substantially impacted by their physical characteristics. Hard particles associate more with the endothelium compared to soft particles, as do small particles compared to large particles, and spherical particles compared to rod‐shaped particles. Transport across the BBB also exhibited a dependence on nanoparticle properties. A nonmonotonic dependence on size was observed, where 200 nm particles exhibited higher BBB transport compared to 100 and 500 nm spheres. Rod‐shaped particles exhibited higher BBB transport when normalized by endothelial association and soft particles exhibited comparable transport to hard particles when normalized by endothelial association. Tuning nanoparticles' physical parameters could potentially enhance their ability to cross the BBB for therapeutic applications.

The disease chytridiomycosis, caused by the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), has contributed to global amphibian declines. Bd infects the keratinized epidermal tissue in amphibians and causes hyperkeratosis and excessive skin shedding. In individuals of susceptible species, the regulatory function of the amphibian's skin is disrupted resulting in an electrolyte depletion, osmotic imbalance, and eventually death. Safe and effective treatments for chytridiomycosis are urgently needed to control chytrid fungal infections and stabilize populations of endangered amphibian species in captivity and in the wild. Currently, the most widely used anti-Bd treatment is itraconazole. Preparations of itraconazole formulated for amphibian use has proved effective, but treatment involves short baths over seven to ten days, a process which is logistically challenging, stressful, and causes long-term health effects. Here, we explore a novel anti-fungal therapeutic using a single application of the ionic liquid, 1-Butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide (BMP-NTf2), for the treatment of chytridiomycosis. BMP-NTf2 was found be effective at killing Bd in vitro at low concentrations (1:1000 dilution). We tested BMP-NTf2 in vivo on two amphibian species, one that is relatively tolerant of chytridiomycosis (Pseudacris regilla) and one that is highly susceptible (Dendrobates tinctorius). A toxicity trial revealed a surprising interaction between Bd infection status and the impact of BMP-NTf2 on D. tinctorius survival. Uninfected D. tinctorius tolerated BMP-NTf2 (mean ± SE; 96.01 ± 9.00 μl/g), such that only 1 out of 30 frogs died following treatment (at a dose of 156.95 μL/g), whereas, a lower dose (mean ± SE; 97.45 ± 3.52 μL/g) was not tolerated by Bd-infected D. tinctorius, where 15 of 23 frogs died shortly upon BMP-NTf2 application. Those that tolerated the BMP-NTf2 application did not exhibit Bd clearance. Thus, BMP-NTf2 application, under the conditions tested here, is not a suitable option for clearing Bd infection in D. tinctorius. However, different results were obtained for P. regilla. Two topical applications of BMP-NTf2 on Bd-infected P. regilla (using a lower BMP-NTf2 dose than on D. tinctorius, mean ± SE; 9.42 ± 1.43 μL/g) reduced Bd growth, although the effect was lower than that obtained by daily doses of itracanozole (50% frogs exhibited complete clearance on day 16 vs. 100% for itracanozole). Our findings suggest that BMP-NTf2 has the potential to treat Bd infection, however the effect depends on several parameters. Further optimization of dose and schedule are needed before BMP-NTf2 can be considered as a safe and effective alternative to more conventional antifungal agents, such as itraconazole.

Ratiometric delivery of combination chemotherapy can achieve therapeutic efficacy based on synergistic interactions between drugs. It is critical to design such combinations with drugs that complement each other and reduce cancer growth through multiple mechanisms. Using hyaluronic acid (HA) as a carrier, two chemotherapeutic agents—doxorubicin (DOX) and camptothecin (CPT)—were incorporated and tested for their synergistic potency against a broad panel of blood-cancer cell lines. The pair also demonstrated the ability to achieve immunogenic cell death by increasing the surface exposure levels of Calreticulin, thereby highlighting its ability to induce apoptosis via an alternate pathway. Global proteomic profiling of cancer cells treated with HA–DOX–CPT identified pathways that could potentially predict patient sensitivity to HA–DOX–CPT. This lays the foundation for further exploration of integrating drug delivery and proteomics in personalized immunogenic chemotherapy.

Unwanted uptake in the liver and limited accumulation in target organs is a major obstacle to targeted drug delivery. Here, the authors report on the hitchhiking of nanocarriers on red blood cells and the targeted upstream delivery to different target organs in mice, pigs and ex vivo human lungs.

Abstract Nanoparticle drug delivery systems have been used in the clinic since the early 1990's. Since that time, the field of nanomedicine has evolved alongside growing technological needs to improve the delivery of various therapeutics. Over these past decades, newer generations of nanoparticles have emerged that are capable of performing additional delivery functions that can enable treatment via new therapeutic modalities. In the current clinical landscape, many of these new generation nanoparticles have reached clinical trials and have been approved for various indications. In the first issue of Bioengineering & Translational Medicine in 2016, we reviewed the history, current clinical landscape, and clinical challenges of nanoparticle delivery systems. Here, we provide a 3 year update on the current clinical landscape of nanoparticle drug delivery systems and highlight newly approved nanomedicines, provide a status update on previous clinical trials, and highlight new technologies that have recently entered the clinic.

Abstract Microfluidic cellular models, commonly referred to as “organs‐on‐chips,” continue to advance the field of bioengineering via the development of accurate and higher throughput models, captivating the essence of living human organs. This class of models can mimic key in vivo features, including shear stresses and cellular architectures, in ways that cannot be realized by traditional two‐dimensional in vitro models. Despite such progress, current organ‐on‐a‐chip models are often overly complex, require highly specialized setups and equipment, and lack the ability to easily ascertain temporal and spatial differences in the transport kinetics of compounds translocating across cellular barriers. To address this challenge, we report the development of a three‐dimensional human blood brain barrier (BBB) microfluidic model (μHuB) using human cerebral microvascular endothelial cells (hCMEC/D3) and primary human astrocytes within a commercially available microfluidic platform. Within μHuB, hCMEC/D3 monolayers withstood physiologically relevant shear stresses (2.73 dyn/cm2) over a period of 24 hr and formed a complete inner lumen, resembling in vivo blood capillaries. Monolayers within μHuB expressed phenotypical tight junction markers (Claudin‐5 and ZO‐1), which increased expression after the presence of hemodynamic‐like shear stress. Negligible cell injury was observed when the monolayers were cultured statically, conditioned to shear stress, and subjected to nonfluorescent dextran (70 kDa) transport studies. μHuB experienced size‐selective permeability of 10 and 70 kDa dextrans similar to other BBB models. However, with the ability to probe temporal and spatial evolution of solute distribution, μHuBs possess the ability to capture the true variability in permeability across a cellular monolayer over time and allow for evaluation of the full breadth of permeabilities that would otherwise be lost using traditional end‐point sampling techniques. Overall, the μHuB platform provides a simplified, easy‐to‐use model to further investigate the complexities of the human BBB in real‐time and can be readily adapted to incorporate additional cell types of the neurovascular unit and beyond.

Abstract Combination chemotherapy is often employed to improve therapeutic efficacies of drugs. However, traditional combination regimens often utilize drugs at or near‐their maximum tolerated doses (MTDs), elevating the risk of dose‐related toxicity and impeding their clinical success. Further, high doses of adjuvant or neoadjuvant chemotherapies can cause myeloablation, which compromises the immune response and hinders the efficacy of chemotherapy as well as accompanying treatments such as immunotherapy. Clinical outcomes can be improved if chemotherapy combinations are designed to reduce the overall doses without compromising their therapeutic efficacy. To this end, we investigated a combination of camptothecin (CPT) with doxorubicin (DOX) as a low‐dose treatment option for breast cancer. DOX‐CPT combinations were synergistic in several breast cancer cell lines in vitro and one particular ratio displayed extremely high synergy on human triple negative breast cancer cells (MDA‐MB‐231). This combination led to excellent long‐term survival of mice bearing MDA‐MB‐231 tumors at doses roughly five‐fold lower than the reported MTD values of its constituent drugs. Impact of low dose DOX‐CPT treatment on local tumor immune environment was assessed in immunocompetent mice bearing breast cancer (4T1) tumors. The combination was not only superior in inhibiting the disease progression compared to individual drugs, but it also generated a more favorable antitumor immunogenic response. Engineering DOX and CPT ratios to manifest synergy enables treatment at doses much lower than their MTDs, which could ultimately facilitate their translation into the clinic as a promising combination for breast cancer treatment.

Liposome-based drug delivery systems have allowed for better drug tolerability and longer circulation times but are often optimized for a single agent due to the inherent difficulty of co-encapsulating two drugs with differing chemical profiles. Here, we design and test a prodrug based on a ribosylated nucleoside form of 5-fluorouracil, 5-fluorouridine (5FUR), with the final purpose of co-encapsulation with doxorubicin (DOX) in liposomes. To improve the loading of 5FUR, we developed two 5FUR prodrugs that involved the conjugation of either one or three moieties of tryptophan (W) known respectively as, 5FUR−W and 5FUR−W3. 5FUR−W demonstrated greater chemical stability than 5FUR−W3 and allowed for improved loading with fewer possible byproducts from tryptophan hydrolysis. Varied drug ratios of 5FUR−W: DOX were encapsulated for in vivo testing in the highly aggressive 4T1 murine breast cancer model. A liposomal molar ratio of 2.5 5FUR−W: DOX achieved a 62.6% reduction in tumor size compared to the untreated control group and a 33% reduction compared to clinical doxorubicin liposomes in a proof-of-concept study to demonstrate the viability of the co-encapsulated liposomes. We believe that the new prodrug 5FUR−W demonstrates a prodrug design with clinical translatability by reducing the number of byproducts produced by the hydrolysis of tryptophan, while also allowing for loading flexibility.

Red blood cells (RBC) have great potential as drug delivery systems, capable of producing unprecedented changes in pharmacokinetics, pharmacodynamics, and immunogenicity. Despite this great potential and nearly 50 years of research, it is only recently that RBC-mediated drug delivery has begun to move out of the academic lab and into industrial drug development. RBC loading with drugs can be performed in several ways—either via encapsulation within the RBC or surface coupling, and either ex vivo or in vivo—depending on the intended application. In this review, we briefly summarize currently used technologies for RBC loading/coupling with an eye on how pharmacokinetics is impacted. Additionally, we provide a detailed description of key ADME (absorption, distribution, metabolism, elimination) changes that would be expected for RBC-associated drugs and address unique features of RBC pharmacokinetics. As thorough understanding of pharmacokinetics is critical in successful translation to the clinic, we expect that this review will provide a jumping off point for further investigations into this area.

Red blood cells (RBCs) can be used for vascular delivery of encapsulated or surface-bound drugs and carriers. Coupling to RBC prolongs circulation of nanoparticles (NP, 200 nm spheres, a conventional model of polymeric drug delivery carrier) enabling their transfer to the pulmonary vasculature without provoking overt RBC elimination. However, little is known about more subtle and potentially harmful effects of drugs and drug carriers on RBCs. Here we devised high-throughput in vitro assays to determine the sensitivity of loaded RBCs to osmotic stress and other damaging insults that they may encounter in vivo (e.g. mechanical, oxidative and complement insults). Sensitivity of these tests is inversely proportional to RBC concentration in suspension and our results suggest that mouse RBCs are more sensitive to damaging factors than human RBCs. Loading RBCs by NP at 1:50 ratio did not affect RBCs, while 10-50 fold higher NP load accentuated RBC damage by mechanical, osmotic and oxidative stress. This extensive loading of RBC by NP also leads to RBCs agglutination in buffer; however, addition of albumin diminished this effect. These results provide a template for analyses of the effects of diverse cargoes loaded on carrier RBCs and indicate that: i) RBCs can tolerate carriage of NP at doses providing loading of millions of nanoparticles per microliter of blood; ii) tests using protein-free buffers and mouse RBCs may overestimate adversity that may be encountered in humans.

OBJECTIVES: Caco-2 monolayers are one of the most widely used in vitro models for prediction of intestinal permeability of therapeutic molecules. However, the conventional Caco-2 monolayer model has several drawbacks including labor-intensive culture process, unphysiological growth conditions, lack of reproducibility and limited throughput. Here, we report on the use of 3-day Caco-2 monolayers for assessing permeability of polypeptide drugs. METHODS: The 3-day monolayers were grown in a commercially available transwell set-up, which facilitates rapid development of the Caco-2 monolayers in an intestinal epithelial differentiation mimicking environment. This set-up included use of serum-free medium of defined composition with supplements such as butyric acid, hormones, growth factors, and other metabolites, reported to regulate the differentiation of intestinal epithelial cells in vivo. We measured permeability of 3 different therapeutic polypeptides; insulin, calcitonin, and exenatide across the monolayer. RESULTS: Preliminary validation of the monolayer was carried out by confirming dose-dependent permeation of FITC-insulin and sulforhodamine-B. Transport of insulin, calcitonin, and exenatide measured at different loading concentrations suggests that the permeability values obtained with 3-day cultures resemble more closely the values obtained with ex vivo models compared to permeability values obtained with conventional 21-day cultures. CONCLUSIONS: Short-term 3-day Caco-2 monolayers provide new opportunities for developing reproducible and high-throughput models for screening of therapeutic macromolecules for oral absorption.

Recognition by macrophages is a key process in generating immune response against invading pathogens. Previous studies have focused on recognition of pathogens through surface receptors present on the macrophage's surface. Here, using polymeric particles of different geometries that represent the size and shape range of a variety of bacteria, the importance of target geometry in recognition was investigated. The studies reported here reveal that attachment of particles of different geometries to macrophages exhibits a strong dependence on size and shape. For all sizes and shapes studied, particles possessing the longest dimension in the range of 2-3 microm exhibited highest attachment. This also happens to be the size range of most commonly found bacteria in nature. The surface features of macrophages, in particular the membrane ruffles, might play an important role in this geometry-based target recognition by macrophages. These findings have significant implications in understanding the pathogenicity of bacteria and in designing drug delivery carriers.

An effective approach to separating shaped particles is needed to isolate disease-causing cells for diagnostics or to aid in purifying nonspherical particles in applications ranging from food science to drug delivery. However, the separation of shaped particles is generally challenging, since nonspherical particles can freely rotate and present different faces while being sorted. We experimentally and numerically show that inertial fluid-dynamic effects allow for shape-dependent separation of flowing particles. (Spheres and rods with aspect ratios of 3∶1 and 5∶1 have all been separable.) Particle rotation around a conserved axis following Jeffery orbits is found to be a necessary component in producing different equilibrium positions across the channel that depend on particle rotational diameter. These differences are large enough to enable passive, continuous, high-purity, high-throughput, and shape-based separation downstream. Furthermore, we show that this shape-based separation can be applied to a large range of particle sizes and types, including small, artificially made 3-μm particles as well as bioparticles such as yeast. This practical approach for sorting particles by a previously inaccessible geometric parameter opens up a new capability that should find use in a range of fields.