Biologics now constitute a significant element of available medical treatments. Owing to their clinical and commercial success, biologics are a rapidly growing class and have become a dominant therapeutic modality. Although most of the successful biologics to date are drugs that bear a peptidic backbone, ranging from small peptides to monoclonal antibodies (~500 residues; 150 kDa), new biologic modalities, such as nucleotide-based therapeutics and viral gene therapies, are rapidly maturing towards widespread clinical use. Given the rise of peptides and proteins in the pharmaceutical landscape, tremendous research and development interest exists in developing less-invasive or non-invasive routes for the systemic delivery of biologics, including subcutaneous, transdermal, oral, inhalation, nasal and buccal routes. This Review summarizes the current status, latest updates and future prospects for such delivery of peptides, proteins and other biologics.
Jacob S Brenner, Daniel C Pan, Jacob W Myerson, Oscar A Marcos-Contreras, Carlos H Villa, Priyal Patel, Hugh Hekierski, Shampa Chatterjee, Jian-Qin Tao, Hamideh Parhiz, Kartik Bhamidipati, Thomas G Uhler, Elizabeth D Hood, Raisa Yu Kiseleva, Vladimir S Shuvaev, Tea Shuvaeva, Makan Khoshnejad, Ian Johnston, Jason V Gregory, Joerg Lahann, Tao Wang, Edward Cantu, William M Armstead, Samir Mitragotri, and Vladimir Muzykantov. 2018. “Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude.” Nat Commun, 9, 1, Pp. 2684.Abstract
Drug delivery by nanocarriers (NCs) has long been stymied by dominant liver uptake and limited target organ deposition, even when NCs are targeted using affinity moieties. Here we report a universal solution: red blood cell (RBC)-hitchhiking (RH), in which NCs adsorbed onto the RBCs transfer from RBCs to the first organ downstream of the intravascular injection. RH improves delivery for a wide range of NCs and even viral vectors. For example, RH injected intravenously increases liposome uptake in the first downstream organ, lungs, by ~40-fold compared with free NCs. Intra-carotid artery injection of RH NCs delivers >10% of the injected NC dose to the brain, ~10× higher than that achieved with affinity moieties. Further, RH works in mice, pigs, and ex vivo human lungs without causing RBC or end-organ toxicities. Thus, RH is a clinically translatable platform technology poised to augment drug delivery in acute lung disease, stroke, and several other diseases.
Combination chemotherapy is commonly used to treat late stage cancer; however, treatment is often limited by systemic toxicity. Optimizing drug ratio and schedule can improve drug combination activity and reduce dose to lower toxicity. Here, we identify gemcitabine (GEM) and doxorubicin (DOX) as a synergistic drug pair in vitro for the triple negative breast cancer cell line MDA-MB-231. Drug synergy and caspase activity were increased the most by exposing cells to GEM prior to DOX in vitro. While the combination was more effective than the single drugs at inhibiting MDA-MB-231 growth in vivo, the clear schedule dependence observed in vitro was not observed in vivo. Differences in drug exposure and cellular behavior in vivo compared to in vitro are likely responsible. This study emphasizes the importance in understanding how schedule impacts drug synergy and the need to develop more advanced strategies to translate synergy to the clinic.
Immunomodulation, manipulation of the immune responses towards an antigen, is a promising strategy to treat cancer, infectious diseases, allergies, and autoimmune diseases, among others. Unique features of the skin including the presence of tissue-resident immune cells, ease of access and connectivity to other organs makes it a unique target organ for immunomodulation. In this review, we summarize advances in transdermal delivery of agents for modulating the immune responses for vaccination as well as tolerization. The biological foundation of skin-based immunomodulation and challenges in its implementation are described. Technological approaches aimed at enhancing the delivery of immunomodulatory therapeutics into skin are also discussed in this review. Progress made in the treatment of several specific diseases including cancer, infections and allergy are discussed. Finally, this review discusses some practical considerations and offers some recommendations for future studies in the field of transdermal immunomodulation.
Transdermal delivery of pharmaceuticals using ionic liquids and deep eutectic solvents (DES) has attracted significant interest due to the inherent tunability of the molecules and their capacity to transport large molecules across the skin. Several key properties of DESs including viscosity, miscibility and possible transport enhancement can be controlled through the choice of ions and their ratio in DES. Herein we investigate the effect of cation/anion ratio using Choline and Geranic acid (CAGE) based DES. We synthesized variants of CAGE by controlling the ratio of Choline to Geranic acid over a range of 1:4 to 2:1. Physicochemical properties including viscosity, conductivity and diffusivity were measured. Effect of CAGE on skin permeability was assessed using insulin in ex vivo porcine skin. Each variant was found to have distinct properties, including interionic interactions, viscosity, and conductivity. In addition, the effect of CAGE on stratum corneum lipids, as assessed by FTIR, was dependent on its composition. Transport enhancement was also composition-dependent, as the variants containing excess geranic acid (1:2 and 1:4, but not geranic acid alone) exhibited higher insulin delivery into the dermis compared to other compositions, demonstrating the importance of investigating the effect of ion ratios on drug delivery.
Combination chemotherapy treatments with polymerdrugconjugates(PDCs) have been designed to overcome the limitations of traditional chemotherapy. While there are many significant design challenges associated with engineering a combination therapy with PDCs, several studies have shown therapeutic benefits of incorporating multiple drugs onto a polymeric carrier. Here, we summarize the chemistries and biological performance of different combination PDCs that have been tested in the preclinical setting and offer recommendations for future studies which will provide the necessary information to streamline future translation.
Intravenously injected nanopharmaceuticals, including PEGylated nanoparticles, induce adverse cardiopulmonary reactions in sensitive human subjects, and these reactions are highly reproducible in pigs. Although the underlying mechanisms are poorly understood, roles for both the complement system and reactive macrophages have been implicated. Here, we show the dominance and importance of robust pulmonary intravascular macrophage clearance of nanoparticles in mediating adverse cardiopulmonary distress in pigs irrespective of complement activation. Specifically, we show that delaying particle recognition by macrophages within the first few minutes of injection overcomes adverse reactions in pigs using two independent approaches. First, we changed the particle geometry from a spherical shape (which triggers cardiopulmonary distress) to either rod- or disk-shape morphology. Second, we physically adhered spheres to the surface of erythrocytes. These strategies, which are distinct from commonly leveraged stealth engineering approaches such as nanoparticle surface functionalization with poly(ethylene glycol) and/or immunological modulators, prevent robust macrophage recognition, resulting in the reduction or mitigation of adverse cardiopulmonary distress associated with nanopharmaceutical administration.
The capability to graft synthetic polymers onto the surfaces of live cells offers the potential to manipulate and control their phenotype and underlying cellular processes. Conventional grafting-to strategies for conjugating preformed polymers to cell surfaces are limited by low polymer grafting efficiency. Here we report an alternative grafting-from strategy for directly engineering the surfaces of live yeast and mammalian cells through cell surface-initiated controlled radical polymerization. By developing cytocompatible PET-RAFT (photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization), synthetic polymers with narrow polydispersity (M/M < 1.3) could be obtained at room temperature in 5 minutes. This polymerization strategy enables chain growth to be initiated directly from chain-transfer agents anchored on the surface of live cells using either covalent attachment or non-covalent insertion, while maintaining high cell viability. Compared with conventional grafting-to approaches, these methods significantly improve the efficiency of grafting polymer chains and enable the active manipulation of cellular phenotypes.
Combination chemotherapy is commonly used to treat advanced breast cancer. However, treatment success is often limited due to systemic toxicity. To improve therapeutic efficacy, polymer drug conjugates carrying synergistic pairs of chemotherapy drugs can be used to reduce drug administration dose. Here, we systematically evaluated the effect of temporal scheduling of doxorubicin (DOX) and gemcitabine (GEM) on drug synergy. Hyaluronic acid (HA) drug conjugates with distinct linkers conjugating both DOX and GEM were synthesized to control relative release kinetics of each drug. We show that polymer conjugates that release GEM faster than DOX are more effective at killing triple negative breast cancer cells in vitro. We further show that the optimal dual drug conjugate more effectively inhibits the growth of an aggressive, orthotopic 4T1 tumor model in vivo than free DOX and GEM and the single drug HA conjugates. The dual drug HA conjugate can inhibit 4T1 tumor growth in vivo during treatment through both intravenous and non-local subcutaneous injections. These results emphasize the importance of understanding the effect release rates have on the efficacy of synergistic drug carriers and motivate the use of HA as a delivery platform for multiple cancer types.
Geometry has been considered as one of the important parameters in nanoparticle design because it affects cellular uptake, transport across the physiological barriers, and in vivo distribution. However, only a few studies have been conducted to elucidate the influence of nanoparticle geometry in their in vivo fate after oral administration. This article discloses the effect of nanoparticle shape on transport and absorption in gastrointestinal (GI) tract. Nanorods and nanospheres were prepared and labeled using fluorescence resonance energy transfer molecules to track the in vivo fate of intact nanoparticles accurately. Results demonstrated that nanorods had significantly longer retention time in GI tract compared with nanospheres. Furthermore, nanorods exhibited stronger ability of penetration into space of villi than nanospheres, which is the main reason of longer retention time. In addition, mesenteric lymph transported 1.75% nanorods within 10 h, which was more than that with nanospheres (0.98%). Fluorescent signals arising from nanoparticles were found in the kidney but not in the liver, lung, spleen, or blood, which could be ascribed to low absorption of intact nanoparticles. In conclusion, nanoparticle geometry influences in vivo fate after oral delivery and nanorods should be further investigated for designing oral delivery systems for therapeutic drugs, vaccines, or diagnostic materials.
Diabetes mellitus has become a major public health issue that has almost reached epidemic proportions worldwide. Injectable insulin has been typically utilized for the management of this chronic disease. However, lack of patient compliance with injectable formulations has spurred the development of oral insulin formulations, which although appealing, face several delivery challenges. We have developed novel mucoadhesive intestinal patches, several hundred micrometers in dimension (micropatches) that address the challenges of oral insulin delivery. The micropatches adhere to the intestinal mucosa, release their drug load rapidly within 30 min and are effective in lowering blood glucose levels in vivo. When insulin-loaded micropatches were administered with a permeation enhancer and protease inhibitor, a peak efficacy of 34% drop in blood glucose levels was observed within 3 h. Efficacy further improved to 41% when micropatches were administered in multiple doses. Here, we describe the design of micropatches as an oral insulin formulation and report their in vivo efficacy.
Intestinal patches provide a unique platform for oral delivery of drugs which possess poor oral bioavailability, necessitating their administration by injections. Intestinal patch based devices prevent drug degradation in the gastrointestinal tract, facilitate their intestinal absorption through forming a localized drug depot at the delivery site and provide unidirectional, controlled drug release while preventing luminal drug loss. Consequently, intestinal patch-based devices are being developed for oral delivery of several drugs such as insulin, exenatide, calcitonin, interferon-α, erythropoietin and human granulocyte colony-stimulating factor for the treatment of diabetes, osteoporosis, hepatitis or for chemotherapy. This technology shows promise as a needle-free alternative to injectable drugs that would improve the quality of lives of millions of people requiring chronic administration of injectable drugs.
In vitro and in vivo assessment of safety and efficacy are the essential first steps in developing nanoparticle-based therapeutic systems. However, it is often challenging to use the knowledge gained from in vitro studies to predict the outcome of in vivo studies since the complexity of the in vivo environment, including the existence of flow and a multicellular environment, is often lacking in traditional in vitro models. Here, we describe a microfluidic co-culture model comprising 4T1 breast cancer cells and EA.hy926 endothelial cells under physiological flow conditions and its utilization to assess the penetration of therapeutic nanoparticles from the vascular compartment into a cancerous cell mass. Camptothecin nanocrystals (∼310 nm in length), surface-functionalized with PEG or folic acid, were used as a test nanocarrier. Camptothecin nanocrystals exhibited only superficial penetration into the cancerous cell mass under fluidic conditions, but exhibited cytotoxicity throughout the cancerous cell mass. This likely suggests that superficially penetrated nanocrystals dissolve at the periphery and lead to diffusion of molecular camptothecin deep into the cancerous cell mass. The results indicate the potential of microfluidic co-culture devices to assess nanoparticle-cancerous cell interactions, which are otherwise difficult to study using standard in vitro cultures.
We report the development of sponge Haliclona sp. spicules, referred to as SHS, and its topical application in skin delivery of hydrophilic biomacromolecules, a series of fluorescein isothiocyanate-dextrans (FDs). SHS are silicious oxeas which are sharp-edged and rod-shaped (∼120 μm in length and ∼7 μm in diameter). SHS can physically disrupt skin in a dose-dependent manner and retain within the skin over at least 72 h, which allows sustained skin penetration of hydrophilic biomacromolecules. The magnitude of enhancement of FD delivery into skin induced by SHS treatment was dependent on its molecular weight. Specifically, SHS topical application enhanced FD-10 (MW: 10 kDa) penetration into porcine skin in vitro by 33.09 ± 7.16-fold compared to control group (p < 0.01). SHS dramatically increased the accumulation of FD-10 into and across the dermis by 62.32 ± 13.48-fold compared to the control group (p < 0.01). In vivo experiments performed using BALB/c mice also confirmed the effectiveness of SHS topical application; the skin absorption of FD-10 with SHS topical application was 72.14 ± 48.75-fold (p < 0.05) and 15.39 ± 9.91-fold (p < 0.05) higher than those from the PBS and Dermaroller microneedling, respectively. Further, skin irritation study and transepidermal water loss (TEWL) measurement using guinea pig skin in vivo indicated that skin disruption induced by SHS treatment is self-limited and can be recovered with time and efficiently. SHS can offer a safe, effective, and sustained skin delivery of hydrophilic biomacromolecules and presents a promising platform technology for a wide range of cosmetic and medical applications.
Multiple emulsions have received great interest due to their ability to be used as templates for the production of multicompartment particles for a variety of applications. However, scaling these complex droplets to nanoscale dimensions has been a challenge due to limitations on their fabrication methods. Here, we report the development of oil-in-water-in-oil (O/W/O) double nanoemulsions via a two-step high-energy method and their use as templates for complex nanogels comprised of inner oil droplets encapsulated within a hydrogel matrix. Using a combination of characterization methods, we determine how the properties of the nanogels are controlled by the size, stability, internal morphology, and chemical composition of the nanoemulsion templates from which they are formed. This allows for identification of compositional and emulsification parameters that can be used to optimize the size and oil encapsulation efficiency of the nanogels. Our templating method produces oil-laden nanogels with high oil encapsulation efficiencies and average diameters of 200-300 nm. In addition, we demonstrate the versatility of the system by varying the types of inner oil, the hydrogel chemistry, the amount of inner oil, and the hydrogel network cross-link density. These nontoxic oil-laden nanogels have potential applications in food, pharmaceutical, and cosmetic formulations.
Transdermal delivery of peptides and other biological macromolecules is limited due to skin's inherent low permeability. Here, the authors report the use of a deep eutectic solvent, choline and geranate (CAGE), to enhance topical delivery of proteins such as bovine serum albumin (BSA, molecular weight: ≈66 kDa), ovalbumin (OVA, molecular weight: ≈45 kDa) and insulin (INS, molecular weight: 5.8 kDa). CAGE enhances permeation of BSA, OVA, and insulin into porcine skin ex vivo, penetrating deep into the epidermis and dermis. Studies using tritium-labeled BSA and fluorescein isothiocyanate labeled insulin show significantly enhanced delivery of proteins into and across porcine skin, penetrating the skin in a time-dependent manner. Fourier transform IR spectra of porcine stratum corneum (SC) samples before and after incubation in CAGE show a reduction in peak area attributed to SC lipid content, suggesting lipid extraction from the SC. Circular dichroism confirms that CAGE does not affect insulin's secondary conformation. In vivo studies in rats show that topical application of 10 U insulin dispersed in CAGE (25 U kg insulin dose) leads to a highly significant 40% drop in blood glucose levels in 4 h that is relatively sustained for 12 h. Taken together, these studies demonstrate that CAGE is a promising vehicle for transdermal delivery of therapeutic proteins; specifically, as a noninvasive delivery alternative to injectable insulin for the treatment of diabetes.