Delivery Of Biomacromolecules Research Articles

Nanoetched Stainless Steel Architecture Enhances Cell Uptake of Biomacromolecules and Alters Protein Corona Abundancy.

Nanotexture on biocompatible surfaces promotes cell adhesion and proliferation. High aspect ratio nanoachitecture serves as an ideal interface between implant materials and host cells that is well-suited for localized therapeutic delivery. Despite this potential, nanotextured surfaces have not been widely applied for biomacromolecule delivery. Here, we employed a low-cost, industrially relevant nanoetching process to modify the surface of biocompatible stainless steel 316 (SS316L), creating nanotextured SS316L (NT-SS316L) as a material for intracellular biomacromolecule delivery. As biomacromolecule cargoes are adsorbed to the steel and ultimately would be used in protein-rich environments, we performed serum protein corona analysis on unmodified SS316L and NT-SS316L using tandem mass spectrometry. We observed an increase in proteins associated with cell adhesion on the surface of NT-SS316L compared to that of SS316L, supporting literature reports of enhanced adhesion on nanotextured materials. For delivery to adherent cells, a "hard corona" of model biomacromolecule cargoes including superfolder green fluorescent protein (sfGFP) charge variants, cytochrome c, and siRNA was adsorbed on NT-SS316L to assess delivery. Nanotextured surfaces enhanced cellular biomacromolecule uptake and delivered cytosolic-functional proteins and nucleic acids through energy-dependent endocytosis. Collectively, these findings indicate that NT-SS316L holds potential as a surface modification for implants to achieve localized drug delivery for a variety of biomedical applications.

Mechanism and performance of choline-based ionic liquids in enhancing nasal delivery of glucagon

Proteins and peptides have been increasingly developed as pharmaceuticals owing to their high potency and low side effects. However, their administration routes are confined to injections, such as intra-muscular and intra-venous injections, making patient compliance a challenge. Hence, non-injectable delivery systems are crucial to expanding the clinical use of proteins and peptides. In this context, two choline-based ionic liquids (ILs), namely, choline geranic acid ([Ch][Ger]) and choline citric acid ([Ch][Cit]), have been identified as promising agents for enhancing the permeation and prolonging the retention time of glucagon (GC) after intra-nasal administration. Notably, intra-nasal delivery of GC via ILs (GC/ILs) elicited rapid and smooth reversal of acute hypoglycaemia without leading to rebound hyperglycaemia in type 1 diabetic rats subjected to insulin induction. In addition, ILs could improve the transcellular transport of GC through electrostatic interaction. ILs could also transiently open inter-cellular tight junctions transiently to facilitate the paracellular transport of GC. Safety tests indicated that continuous intra-nasal delivery of ILs led to reversible changes, such as epithelial cell inflammation, goblet cell overgrowth, and impacts on the distribution of nasal cilia. However, these changes could be alleviated by the innate self-repair ability of mucosal epithelial cells. This study highlights the considerable potential of ILs for long-term nasal delivery of biomacromolecules.

Enhanced oral and pulmonary delivery of biomacromolecules via amplified transporter targeting

Ligand-modified nanocarriers can promote oral or inhalative administration of macromolecular drugs across the intestinal or pulmonary mucosa. However, enhancing the unidirectional transport of the nanocarriers through “apical uptake→intracellular transport→basolateral exocytosis” route remains a hot topic and challenge in current research. Forskolin is a naturally occurring diterpenoid compound extracted from the roots of C. forskohlii. In our studies, we found that forskolin could increase the transcellular transport of butyrate-modified nanoparticles by 1.67-fold and 1.20-fold in Caco-2 intestinal epithelial cell models and Calu-3 lung epithelial cell models, respectively. Further mechanistic studies revealed that forskolin, on the one hand, promoted the cellular uptake of butyrate-modified nanoparticles by upregulating the expression of monocarboxylic acid transporter-1 (MCT-1) on the apical membrane. On the other hand, forskolin facilitated the binding of MCT-1 to caveolae, thereby mediating butyrate-modified nanoparticles hijacking caveolae to promote the basolateral exocytosis of butyrate-modified nanoparticles. Studies in normal mice model showed that forskolin could promote the transmucosal absorption of butyrate-modified nanoparticles by >2-fold, regardless of oral or inhalative administration. Using semaglutide as the model drug, both oral and inhalation delivery approaches demonstrated significant hypoglycemic effects in type 2 diabetes mice model, in which inhalative administration was more effective than oral administration. This study optimized the strategies aimed at enhancing the transmucosal absorption of ligand-modified nanocarriers in the intestinal or pulmonary mucosa.

Reversible Assembly of Proteins and Phenolic Polymers for Intracellular Protein Delivery with Serum Stability.

The design of intracellular delivery systems for protein drugs remains a challenge due to limited delivery efficacy and serum stability. Herein, we propose a reversible assembly strategy to assemble cargo proteins and phenolic polymers into stable nanoparticles for this purpose using a heterobifunctional adaptor (2-formylbenzeneboronic acid). The adaptor is easily decorated on cargo proteins via iminoboronate chemistry and further conjugates with catechol-bearing polymers to form nanoparticles via boronate diester linkages. The nanoparticles exhibit excellent serum stability in culture media but rapidly release the cargo proteins triggered by lysosomal acidity and GSH after endocytosis. In a proof-of-concept animal model, the strategy successfully transports superoxide dismutase to retina via intravitreal injection and efficiently ameliorates the oxidative stress and cellular damage in the retina induced by ischemia-reperfusion (I/R) with minimal adverse effects. The reversible assembly strategy represents a robust and efficient method to develop serum-stable systems for the intracellular delivery of biomacromolecules.

Non-invasive transdermal delivery of biomacromolecules with fluorocarbon-modified chitosan for melanoma immunotherapy and viral vaccines

Transdermal drug delivery has been regarded as an alternative to oral delivery and subcutaneous injection. However, needleless transdermal delivery of biomacromolecules remains a challenge. Herein, a transdermal delivery platform based on biocompatible fluorocarbon modified chitosan (FCS) is developed to achieve highly efficient non-invasive delivery of biomacromolecules including antibodies and antigens. The formed nanocomplexes exhibits effective transdermal penetration ability via both intercellular and transappendageal routes. Non-invasive transdermal delivery of immune checkpoint blockade antibodies induces stronger immune responses for melanoma in female mice and reduces systemic toxicity compared to intravenous injection. Moreover, transdermal delivery of a SARS-CoV-2 vaccine in female mice results in comparable humoral immunity as well as improved cellular immunity and immune memory compared to that achieved with subcutaneous vaccine injection. Additionally, FCS-based protein delivery systems demonstrate transdermal ability for rabbit and porcine skins. Thus, FCS-based transdermal delivery systems may provide a compelling opportunity to overcome the skin barrier for efficient transdermal delivery of bio-therapeutics.

Unleashing the Power of Proenzyme Delivery for Targeted Therapeutic Applications Using Biodegradable Lipid Nanoparticles.

Proenzymes, functioning as inactive precursor forms of enzymes, hold significant promise for regulating essential biological processes. Their inherent property of latency, remaining inert until they arrive at the intended site of action, positions them as particularly promising candidates for the development of targeted therapeutics. Despite this potential, the therapeutic potential of proenzymes is challenged by designing proenzymes with excellent selectivity for disease cells. This limitation is further exacerbated by the inability of proenzymes to spontaneously cross the cell membrane, a biological barrier that impedes the cellular internalization of exogenous macromolecules. Therefore, efficacious intracellular delivery is paramount to unlocking the full therapeutic potency of proenzymes.In this Account, we first elucidate our recent advancements made in designing biodegradable lipid nanoparticles (LNPs) for the cell-specific delivery of biomacromolecules, including proteins and nucleic acids. Using a strategy of parallel synthesis, we have constructed an extensive library of ionizable lipids, each integrated with different biodegradable moieties. This combinatorial approach has led to the identification of LNPs that are particularly efficacious for the delivery of biomacromolecules specifically to tumor cells. This innovation capitalizes on the unique intracellular environment of cancer cells to control the degradation of LNPs, thereby ensuring the targeted release of therapeutics within tumor cells. Additionally, we discuss the structure-activity relationship governing the delivery efficacy of these LNPs and their applicability in regulating tumor cell signaling, specifically through the delivery of bacterial effector proteins.In the second segment, we aim to provide an overview of our recent contributions to the field of proenzyme design, where we have chemically tailored proteins to render them responsive to the unique milieu of tumor cells. Specifically, we elaborate on the chemical principles employed to modify proteins and DNAzymes, thereby priming them for activation in the presence of NAD(P)H:quinone oxidoreductase 1 (NQO1), an enzyme that is prevalently upregulated within tumor cells. We summarize the methodologies for intracellular delivery of these proenzymes using biodegradable LNPs, both in vitro and in vivo. The concomitant intracellular delivery and activation of proenzymes are examined in the context of enhanced therapeutic outcomes and targeted CRISPR/Cas9 genome editing.In conclusion, we offer a perspective on the chemical principles that could be leveraged to optimize LNPs for tissue-specific delivery of proenzymes. We also explore chemical strategies for the irreversible modulation of proenzyme activity within living cells and in vivo. Through this discussion, we provide insights into potential avenues for overcoming existing limitations and enhancing the delivery of proenzymes using LNPs, particularly for developing tumor-targeted therapies and genome editing applications.

Alcohol-containing protein transduction domain mimics

The application and design of protein transduction domains (PTDs) and protein transduction domain mimics (PTDMs) have revolutionized the field of biomacromolecule delivery. Our group has previously synthesized block copolymer PTDMs with well-defined hydrophobic and cationic blocks via ring-opening metathesis polymerization (ROMP). We have optimized the balance of hydrophobicity and cationic density to intracellularly deliver model proteins, active proteins, and antibodies. Despite the presence of serine, threonine, and tyrosine in naturally occurring PTDs, synthetic analogs have yet to be studied in PTDMs. In our present work, we introduce different alcohol groups to our PTDM structures as a new design parameter. A library of nine novel PTDMs were synthesized to incorporate alcohol groups of varying structures and evaluated based on their ability to intracellularly deliver fluorescently labeled antibodies. One PTDM in this novel library, named PTDM4, incorporates alcohol groups in both the hydrophobic and cationic blocks and was found to be the best performing PTDM with almost twice the median fluorescence intensity of the delivered antibody and half the cationic density compared to our positive control, a PTDM thoroughly studied by our group. PTDM4 was further studied by intracellularly delivering the active enzyme, TAT-Cre Recombinase. The activity of TAT-Cre Recombinase delivered by PTDM4 was comparable to that of the positive control, again with half the cationic density. This study is one of the first to examine the effects of alcohol groups on intracellular antibody and active enzyme delivery.

UV-assisted punching of microgel shapes for oral biomacromolecule delivery

UV-assisted punching of microgel shapes for oral biomacromolecule delivery

Metal organic frameworks-in-nanochannels: A tailorable chromatography membrane for isolation of target protein

Metal-organic frameworks (MOFs) demonstrate strong potential in biosample separation. However, the obtained MOFs powders are unsuitable for recovery techniques in an aqueous solution, especially the challenges of withdrawing MOFs particles and expanding their functions for specific applications. Herein, a general strategy is designed utilizing metal oxide-nanochannel arrays as precursors and templates for in-situ selective growth of MOFs structures. The exemplary MOFs (Ni-bipy) with tailored composition are selectively grown in NiO/TiO2 nanochannel membrane (NM) using NiO as the sacrificial precursor, which enables one to achieve a ∼262 times concentration of histidine-tagged proteins within 100 min. The significantly improved adsorption efficiency in a wide pH range and the effective enrichment from a complex matrix as a nanofilter illustrate the great potential of MOFs in nanochannels membranes for the high-efficiency recovery of essential proteins in complex biological samples. The porous self-aligned Ni-MOFs/TiO2 NM exhibits biocompatibility and flexible functionalities, which is desirable for the generation of multifunctional nanofilter devices and developing biomacromolecule delivery vehicles.

Oral Delivery of Protein Drugs via Lysine Polymer-Based Nanoparticle Platforms.

Oral delivery of proteins has opened a new perspective for the treatment of different diseases. However, advances of oral protein formulation are usually hindered by protein susceptibility and suboptimal absorption in the gastrointestinal tract (GIT). Polymeric nano drug delivery systems are considered revolutionary candidates to solve these issues, which can be preferably tunable against specific delivery challenges. Herein, a tailored family of lysine-based poly(ester amide)s (Lys-aaPEAs) is designed as a general oral protein delivery platform for efficient protein loading and protection from degradation. Insulin, as a model protein, can achieve effective internalization by epithelial cells and efficient transport across the intestinal epithelium layer into the systemic circulation, followed by controlled release in physiological environments. After the oral administration of insulin carried by Lys-aaPEAs with ornamental hyaluronic acid (HA), mice with type 1 diabetes mellitus showed an acceptable hypoglycemic effect with alleviated complications. A successful oral insulin delivery is associated with patient comfort and convenience and simultaneously avoids the risk of hypoglycemia compared with injections, which is of great feasibility for daily diabetes therapy. More importantly, this versatile Lys-aaPEAs polymeric library can be recognized as a universal vehicle for oral biomacromolecule delivery, providing more possibilities for treating various diseases.

Overcoming barriers to patient adherence: the case for developing innovative drug delivery systems.

Poor medication adherence is a pervasive issue with considerable health and socioeconomic consequences. Although the underlying reasons are generally understood, traditional intervention strategies rooted in patient-centric education and empowerment have proved to be prohibitively complex and/or ineffective. Formulating a pharmaceutical in a drug delivery system (DDS) is a promising alternative that can directly mitigate many common impediments to adherence, including frequent dosing, adverse effects and a delayed onset of action. Existing DDSs have already positively influenced patient acceptability and improved rates of adherence across various disease and intervention types. The next generation of systems have the potential to instate an even more radical paradigm shift by, for example, permitting oral delivery of biomacromolecules, allowing for autonomous dose regulation and enabling several doses to be mimicked with a single administration. Their success, however, iscontingent on their ability to address the problems that have made DDSs unsuccessful in the past.

Ionic Liquids: Momentous Tools in Transdermal Delivery of Biomacromolecules

AbstractTransdermal drug delivery is a promising strategy characterized by minor fluctuations in blood concentrations, few adverse effects, convenience, and excellent patient compliance, especially for biomacromolecules. Conversely, traditional delivery methods are limited by their poor penetration abilities and complex preparation and application techniques. However, some inherent properties of biomacromolecules, such as large sizes, complex structure, and poor stability, can affect their ability to overcome skin barriers and reach deeper layers. Ionic liquids (ILs) are organic salts composed of asymmetric cations and anions in liquid form below 100 °C. ILs provide excellent protection for biomacromolecules and can facilitate their passage through the stratum corneum to cells for therapeutic effects. Therefore, they represent one of the most promising approaches to promoting transdermal penetration. Here, the barriers to effective transdermal delivery, including skin properties and the stability and biocompatibility of IL‐based biomacromolecule formulations, are highlighted and the current status of IL‐based biomacromolecule formulations are focused on, the tremendous advantages of using ILs based on current challenges in transdermal drug delivery are discussed, and light is shed on several ILs frequently used in the delivery of biomacromolecules.

Hyaluronic-acid based redox-responsive hydrogels using the Diels-Alder reaction for on-demand release of biomacromolecules

Hydrogels are crosslinked, three-dimensional hydrophilic polymeric networks which possess similarity in structure to human soft tissue, and thus have found a great number of applications as biomaterials. Hydrogels can also act as depot for therapeutic cargo, where small molecule drugs to biomolecules can be encapsulated within their porous structure. The aim of this work is to fabricate redox-responsive hydrogels that can dissolve and release biomacromolecules upon exposure to a reducing environment. In particular, hyaluronic acid (HA), was modified with electron-rich furan groups to yield hydrogels through the Diels-Alder (DA) reaction with a maleimide group containing redox-responsive polyethylene glycol (PEG) based crosslinker. Redox-responsive hydrogels were obtained with high conversions (87–95%) at 37 °C, in a catalyst-free fashion. The stimuli-responsive degradation of hydrogels could be tuned by varying the amount of crosslinker. To examine the passive and on-demand delivery of biomacromolecules, a model protein, fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA), was encapsulated within the hydrogels. While a sustained passive release was observed in PBS, a rapid on-demand release was obtained upon exposure to the reducing agent 1,4-dithiothreitol (DTT). Furthermore, the complete dissolution of the hydrogel was accomplished upon exposure to a solution containing the reducing agent.

A Cu+ /Thiourea Dendrimer Achieves Excellent Cytosolic Protein Delivery via Enhanced Cell Uptake and Endosome Escape.

Intracellular protein delivery has attracted considerable attention in the development of protein-based therapeutics, however, the design of highly efficient materials for robust delivery of native proteins remains challenging. This study proposes a Cu+ -based coordination polymer for cytosolic protein delivery with high efficacy and robustness. The phenylthiourea grafted dendrimer is coordinated with cuprous ions to prepare the polymeric carrier, which efficiently bind cargo proteins via a combination of coordination, ionic and hydrophobic interactions. The incorporation of Cu+ ions in the polymer greatly improves its cellular uptake and endosomal escape. The cuprous-based coordination polymer successfully delivered a variety of structurally diverse proteins into various cell lines with reserved bioactivities. This study provides a new type of coordination polymers for cytosolic delivery of biomacromolecules.

Engineered bacteria combined with doxorubicin nanoparticles suppress angiogenesis and metastasis in murine melanoma models.

Methods capable of distributing antitumour therapeutics uniformly throughout an entire tumour and that can suppress metastasis at the same time, would be of great significance in improving cancer treatment. Bacteria-mediated synergistic therapies have been explored for better specificity, temporal and spatial controllability, as well for providing regulation of the immune microenvironment, in order to provide improved cancer treatment. To achieve this goal, here we developed an engineered bacteria delivery system (GDOX@HSEc) using synthetic biology and interfacial chemistry technologies. The engineered bacteria were concurrently modified to express heparin sulfatase 1 (HSulf-1) inside (HSEc), to attach doxorubicin-loaded glycogen nanoparticles (GDOX NPs) on their surface. Here we demonstrate that HSEc can actively target and colonise tumour sites resulting in HSulf-1 overexpression, thereby suppressing angiogenesis and metastasis. Simultaneously, the GDOX NPs were able to penetrate into tumour cells, leading to intracellular DNA damage. Our results confirmed that a combination of biotherapy and chemotherapy using GDOX@HSEc resulted in significant melanoma suppression in murine models, with reduced side effects. This study provides a powerful platform for the simultaneous delivery of biomacromolecules and chemotherapeutic drugs to tumours, representing an innovative strategy potentially more effective in treating solid tumours. STATEMENT OF SIGNIFICANCE: An original engineered bacteria-based system (GDOX@HSEc) was developed using synthetic biology and interfacial chemistry technologies to concurrently produce naturally occurring heparin sulfatase 1 (HSulf-1) inside and anchor doxorubicin-loaded glycogen nanoparticles on the surface. GDOX@HSEc allowed for combined local delivery of chemotherapeutic agents along with the enzymes and immunostimulatory bacterial adjuvants, which resulted in a synergistic action in the inhibition of tumour growth and metastasis. The study provides a potential therapeutic approach that allows therapeutic agents to be distributed in a spatiotemporally controllable manner in tumours for combinatorial enhanced therapy.

Discovery of endosomalytic cell-penetrating peptides based on bacterial membrane-targeting sequences

Cell-penetrating peptides (CPPs) are prominent scaffolds for drug developments and related research, particularly the endocytic delivery of biomacromolecules. Effective cargo release from endosomes prior to lysosomal degradation is a crucial step, where the rational design and selection of CPPs remains a challenge and calls for deeper mechanistic understandings. Here, we have investigated a strategy of designing CPPs that selectively disrupt endosomal membranes based on bacterial membrane targeting sequences (MTSs). Six synthesized MTS peptides all exhibit cell-penetrating abilities, among which two d-peptides (d-EcMTS and d-TpMTS) are able to escape from endosomes and localize at ER after entering the cell. The utility of this strategy has been demonstrated by the intracellular delivery of green fluorescent protein (GFP). Together, these results suggest that the large pool of bacterial MTSs may be a rich source for the development of novel CPPs.

Confining the Sol‐Gel Reaction at the Water/Oil Interface: Creating Compartmentalized Enzymatic Nano‐Organelles for Artificial Cells

AbstractLiving organisms compartmentalize their catalytic reactions in membranes for increased efficiency and selectivity. To mimic the organelles of eukaryotic cells, we develop a mild approach for in situ encapsulating enzymes in aqueous‐core silica nanocapsules. In order to confine the sol‐gel reaction at the water/oil interface of miniemulsion, we introduce an aminosilane to the silica precursors, which serves as both catalyst and an amphiphilic anchor that electrostatically assembles with negatively charged hydrolyzed alkoxysilanes at the interface. The semi‐permeable shell protects enzymes from proteolytic attack, and allows the transport of reactants and products. The enzyme‐carrying nanocapsules, as synthetic nano‐organelles, are able to perform cascade reactions when enveloped in a polymer vesicle, mimicking the hierarchically compartmentalized reactions in eukaryotic cells. This in situ encapsulation approach provides a versatile platform for the delivery of biomacromolecules.

Confining the Sol-Gel Reaction at the Water/Oil Interface: Creating Compartmentalized Enzymatic Nano-Organelles for Artificial Cells.

Living organisms compartmentalize their catalytic reactions in membranes for increased efficiency and selectivity. To mimic the organelles of eukaryotic cells, we develop a mild approach for in situ encapsulating enzymes in aqueous-core silica nanocapsules. In order to confine the sol-gel reaction at the water/oil interface of miniemulsion, we introduce an aminosilane to the silica precursors, which serves as both catalyst and an amphiphilic anchor that electrostatically assembles with negatively charged hydrolyzed alkoxysilanes at the interface. The semi-permeable shell protects enzymes from proteolytic attack, and allows the transport of reactants and products. The enzyme-carrying nanocapsules, as synthetic nano-organelles, are able to perform cascade reactions when enveloped in a polymer vesicle, mimicking the hierarchically compartmentalized reactions in eukaryotic cells. This in situ encapsulation approach provides a versatile platform for the delivery of biomacromolecules.

Functionalized PAMAM constructed nanosystems for biomacromolecule delivery.

Polyamidoamines (PAMAMs) are a class of dendrimer with monodispersity and controlled topology, which can deliver biologically active macromolecules (e.g., genes and proteins) to specific regions with high efficiency and minimum side effects. In detail, PAMAMs can be functionalized easily by core modification or surface amendment to encapsulate a wide range of biomacromolecules. Besides, self-assembled, cross-linked and hybrid PAMAMs with customized therapeutic purposes are developed as delivery vehicles, which makes PAMAMs promising for biomacromolecule therapy. In this review, we comprehensively summarize the application of PAMAMs in biomacromolecule delivery from the synthesis of functionalized PAMAM carriers to the development of PAMAM-based drug delivery systems. The underlying strategies for PAMAM functionalization and assembly are first systematically discussed, and then the current applications of PAMAMs for biomacromolecule delivery are reviewed. Finally, a brief perspective on the further applications of PAMAMs concludes, aiming to provide insights into developing PAMAM-based biomacromolecule delivery systems.

Plant biomacromolecule delivery methods in the 21st century.

The 21st century witnessed a boom in plant genomics and gene characterization studies through RNA interference and site-directed mutagenesis. Specifically, the last 15 years marked a rapid increase in discovering and implementing different genome editing techniques. Methods to deliver gene editing reagents have also attempted to keep pace with the discovery and implementation of gene editing tools in plants. As a result, various transient/stable, quick/lengthy, expensive (requiring specialized equipment)/inexpensive, and versatile/specific (species, developmental stage, or tissue) methods were developed. A brief account of these methods with emphasis on recent developments is provided in this review article. Additionally, the strengths and limitations of each method are listed to allow the reader to select the most appropriate method for their specific studies. Finally, a perspective for future developments and needs in this research area is presented.