Regulation Of Cellular Behavior Research Articles

Deformable microlaser force sensing

Mechanical forces are key regulators of cellular behavior and function, affecting many fundamental biological processes such as cell migration, embryogenesis, immunological responses, and pathological states. Specialized force sensors and imaging techniques have been developed to quantify these otherwise invisible forces in single cells and in vivo. However, current techniques rely heavily on high-resolution microscopy and do not allow interrogation of optically dense tissue, reducing their application to 2D cell cultures and highly transparent biological tissue. Here, we introduce DEFORM, deformable microlaser force sensing, a spectroscopic technique that detects sub-nanonewton forces with unprecedented spatio-temporal resolution. DEFORM is based on the spectral analysis of laser emission from dye-doped oil microdroplets and uses the force-induced lifting of laser mode degeneracy in these droplets to detect nanometer deformations. Following validation by atomic force microscopy and development of a model that links changes in laser spectrum to applied force, DEFORM is used to measure forces in 3D and at depths of hundreds of microns within tumor spheroids and late-stage Drosophila larva. We furthermore show continuous force sensing with single-cell spatial and millisecond temporal resolution, thus paving the way for non-invasive studies of biomechanical forces in advanced stages of embryogenesis, tissue remodeling, and tumor invasion.

Scaling up of a Self-Confined Catalytic Hybridization Circuit for Robust microRNA Imaging.

The precise regulation of cellular behaviors within a confined, crowded intracellular environment is highly amenable in diagnostics and therapeutics. While synthetic circuitry system through a concatenated chemical reaction network has rarely been reported to mimic dynamic self-assembly system. Herein, a catalytic self-defined circuit (CSC) for the hierarchically concatenated assembly of DNA domino nanostructures is engineered. By incorporating pre-sealed symmetrical fragments into the preying hairpin reactants, the CSC system allows the hierarchical DNA self-assembly via a microRNA (miRNA)-powered self-sorting catalytic hybridization reaction. With minimal strand complexity, this self-sustainable CSC system streamlined the circuit component and achieved localization-intensified cascaded signal amplification. Profiting from the self-adaptively concatenated hybridization reaction, a reliable and robust method has been achieved for discriminating carcinoma tissues from the corresponding para-carcinoma tissues. The CSC-sustained self-assembly strategy provides a comprehensive and smart toolbox for organizing various hierarchical DNA nanostructures, which may facilitate more insights for clinical diagnosis and therapeutic assessment.

Roles of organokines in intervertebral disc homeostasis and degeneration.

The intervertebral disc is not isolated from other tissues. Recently, abundant research has linked intervertebral disc homeostasis and degeneration to various systemic diseases, including obesity, metabolic syndrome, and diabetes. Organokines are a group of diverse factors named for the tissue of origin, including adipokines, osteokines, myokines, cardiokines, gastrointestinal hormones, and hepatokines. Through endocrine, paracrine, and autocrine mechanisms, organokines modulate energy homeostasis, oxidative stress, and metabolic balance in various tissues to mediate cross-organ communication. These molecules are involved in the regulation of cellular behavior, inflammation, and matrix metabolism under physiological and pathological conditions. In this review, we aimed to summarize the impact of organokines on disc homeostasis and degeneration and the underlying signaling mechanism. We focused on the regulatory mechanisms of organokines to provide a basis for the development of early diagnostic and therapeutic strategies for disc degeneration.

Lung Inflammatory Phenotype in Mice Deficient in Fibulin-2 and ADAMTS-12.

Interaction between extracellular matrix (ECM) components plays an important role in the regulation of cellular behavior and hence in tissue function. Consequently, characterization of new interactions within ECM opens the possibility of studying not only the functional but also the pathological consequences derived from those interactions. We have previously described the interaction between fibulin2 and ADAMTS-12 in vitro and the effects of that interaction using cellular models of cancer. Now, we generate a mouse deficient in both ECM components and evaluate functional consequences of their absence using different cancer and inflammation murine models. The main findings indicate that mice deficient in both fibulin2 and ADAMTS12 markedly increase the development of lung tumors following intraperitoneal urethane injections. Moreover, inflammatory phenotype is exacerbated in the lung after LPS treatment as can be inferred from the accumulation of active immune cells in lung parenchyma. Overall, our results suggest that protective effects in cancer or inflammation shown by fibulin2 and ADAMTS12 as interactive partners in vitro are also shown in a more realistic in vivo context.

Computational evaluation of light propagation in cylindrical bioreactors for optogenetic mammalian cell cultures.

Light-inducible regulation of cellular pathways and gene circuits in mammalian cells is a new frontier in mammalian genetic engineering. Optogenetic mammalian cell cultures, which are light-sensitive engineered cells, utilize light to regulate gene expression and protein activity. As a low-cost, tunable, and reversible input, light is highly adept at spatiotemporal and orthogonal regulation of cellular behavior. However, light is absorbed and scattered as it travels through media and cells, and the applicability of optogenetics in larger mammalian bioreactors has not been determined. In this work, we computationally explore the size limit to which optogenetics can be applied in cylindrical bioreactors at relevant height-to-diameter ratios. We model the propagation of light using the radiative transfer equation and consider changes in reactor volume, absorption coefficient, scattering coefficient, and scattering anisotropy. We observe sufficient light penetration for activation in simulated bioreactors with sizes of up to 80,000 L at maximal cell densities. We performed supporting experiments and found that significant attenuation occurs at the boundaries of the system, but the relative change in intensity distribution within the reactor was consistent with simulation results. We conclude that optogenetics can be applied to bioreactors at an industrial scale and may be a valuable tool for specific biomanufacturing applications.

Telomerase-Mediated Self-Assembly of DNA Network in Cancer Cells Enabling Mitochondrial Interference.

The precise control of the artificially induced reactions inside living cells is emerging as an effective strategy for the regulation of cell functions. Nevertheless, the manipulation of the assembly of exogenous molecules into artificial architectures in response to intracellular-specific signals remains a grand challenge. Herein, we achieve the precise self-assembly of deoxyribonucleic acid (DNA) network inside cancer cells, specifically responding to telomerase, and realize effective mitochondrial interference and the consequent regulation of cellular behaviors. Two functional DNA modules were designed: a mitochondria-targeting branched DNA and a telomerase-responsive linear DNA. Upon uptake by cancer cells, the telomerase primer in linear DNA responded to telomerase, and a strand displacement reaction was triggered by the reverse transcription of telomerase, thus releasing a linker DNA from the linear DNA. The linker DNA afterward hybridized with the branched DNA to form a DNA network on mitochondria. The DNA network interfered with the function of mitochondria, realizing the apoptosis of cancer cells. This system was further administered in a nude mouse tumor model, showing remarkable suppression of tumor growth. We envision that the telomerase-mediated intracellular self-assembly of the DNA network provides a promising route for cancer therapy.

3D Printed Magnet‐Infused Origami Platform for 3D Cell Culture Assessments

Abstract3D printed platforms have diverse possible applications in cell‐based assays, creating biomimetic tissue or organ microenvironments, delineating cell‐to‐cell, cell‐to‐matrix, or cell‐to‐local microenvironment interactions, and investigating drug discovery. Existing engineering techniques limit physiologically relevant 3D cell proliferation and spatiotemporal organization of cells. Herein, a facile fabrication strategy is proposed for magnetically controllable, shape‐morphing, superparamagnetic 3D iron oxide nanoparticle/cellulose acetate scaffolds produced via origami‐inspired 3D printing technology. The additional dimensionality allows the creation of highly customizable 3D in vitro magnetoactive cell culture platforms in which cells can experience gravity with uniform surface morphology, favorable long‐term biodegradability, and low iron ion release that can be actuated numerous times. Leveraging this platform's properties, the spatial and temporal proliferation of Saccharomyces cerevisiae cells are demonstrated, enabling pre‐ and post‐folding dynamic regulation of cellular behaviors at a local level. It is further demonstrated that the viability of the seeded NIH/3T3 fibroblasts remains > 94% and spreads and proliferates within the scaffold channels over a period of 7 d in culture. Therefore, this transformative cell culture assessment could provide alternative directions to revolutionize the 3D cell culture platform to monitor cellular responses to drugs, compounds, and external stimuli and advance personalized treatments.

Differential trafficking of ligands trogocytosed via CD28 versus CTLA4 promotes collective cellular control of co-stimulation

Intercellular communication is crucial for collective regulation of cellular behaviors. While clustering T cells have been shown to mutually control the production of key communication signals, it is unclear whether they also jointly regulate their availability and degradation. Here we use newly developed reporter systems, bioinformatic analyses, protein structure modeling and genetic perturbations to assess this. We find that T cells utilize trogocytosis by competing antagonistic receptors to differentially control the abundance of immunoregulatory ligands. Specifically, ligands trogocytosed via CD28 are shuttled to the T cell surface, enabling them to co-stimulate neighboring T cells. In contrast, CTLA4-mediated trogocytosis targets ligands for degradation. Mechanistically, this fate separation is controlled by different acid-sensitivities of receptor-ligand interactions and by the receptor intracellular domains. The ability of CD28 and CTLA4 to confer different fates to trogocytosed ligands reveals an additional layer of collective regulation of cellular behaviors and promotes the robustness of population dynamics.

Recent Advances in Macroporous Hydrogels for Cell Behavior and Tissue Engineering.

Hydrogels have been extensively used as scaffolds in tissue engineering for cell adhesion, proliferation, migration, and differentiation because of their high-water content and biocompatibility similarity to the extracellular matrix. However, submicron or nanosized pore networks within hydrogels severely limit cell survival and tissue regeneration. In recent years, the application of macroporous hydrogels in tissue engineering has received considerable attention. The macroporous structure not only facilitates nutrient transportation and metabolite discharge but also provides more space for cell behavior and tissue formation. Several strategies for creating and functionalizing macroporous hydrogels have been reported. This review began with an overview of the advantages and challenges of macroporous hydrogels in the regulation of cellular behavior. In addition, advanced methods for the preparation of macroporous hydrogels to modulate cellular behavior were discussed. Finally, future research in related fields was discussed.

On modeling the multiscale mechanobiology of soft tissues: Challenges and progress.

Tissues grow and remodel in response to mechanical cues, extracellular and intracellular signals experienced through various biological events, from the developing embryo to disease and aging. The macroscale response of soft tissues is typically nonlinear, viscoelastic anisotropic, and often emerges from the hierarchical structure of tissues, primarily their biopolymer fiber networks at the microscale. The adaptation to mechanical cues is likewise a multiscale phenomenon. Cell mechanobiology, the ability of cells to transform mechanical inputs into chemical signaling inside the cell, and subsequent regulation of cellular behavior through intra- and inter-cellular signaling networks, is the key coupling at the microscale between the mechanical cues and the mechanical adaptation seen macroscopically. To fully understand mechanics of tissues in growth and remodeling as observed at the tissue level, multiscale models of tissue mechanobiology are essential. In this review, we summarize the state-of-the art modeling tools of soft tissues at both scales, the tissue level response, and the cell scale mechanobiology models. To help the interested reader become more familiar with these modeling frameworks, we also show representative examples. Our aim here is to bring together scientists from different disciplines and enable the future leap in multiscale modeling of tissue mechanobiology.

Insights in the Complex DegU, DegS, and Spo0A Regulation System of Paenibacillus polymyxa by CRISPR-Cas9-Based Targeted Point Mutations.

ABSTRACTDespite being unicellular organisms, bacteria undergo complex regulation mechanisms which coordinate different physiological traits. Among others, DegU, DegS, and Spo0A are the pleiotropic proteins which govern various cellular responses and behaviors. However, the functions and regulatory networks between these three proteins are rarely described in the highly interesting bacterium Paenibacillus polymyxa. In this study, we investigate the roles of DegU, DegS, and Spo0A by introduction of targeted point mutations facilitated by a CRISPR-Cas9-based system. In total, five different mutant strains were generated, the single mutants DegU Q218*, DegS L99F, and Spo0A A257V, the double mutant DegU Q218* DegS L99F, and the triple mutant DegU Q218* DegS L99F Spo0A A257V. Characterization of the wild-type and the engineered strains revealed differences in swarming behavior, conjugation efficiency, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed a significant increase in conjugation efficiency as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences in the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights into the complex regulatory system of P. polymyxa DSM 365.IMPORTANCE To date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa. In this study, we investigate several regulatory proteins which play a role in governing different physiological traits. Precise targeted point mutations were introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, to the model bacterium Bacillus subtilis with regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance over the wild-type. The applicability of the CRISPR-Cas9 system as a robust genome editing tool, in combination with the engineered strain with increased genetic accessibility, would boost further research in P. polymyxa and support its utilization for biotechnological applications. Overall, our study provides novel insights, which will be of importance in understanding how multiple cellular processes are regulated in Paenibacillus species.

Alternative Splicing in Cancer and Immune Cells.

Simple SummaryAlternative splicing is one of the most fabulous and important mechanism in the cell. Alternative splicing is capable of generating many proteins from a single gene and can be involved in many pathways. In this review, we decided to present a review of the literature on alternative splicing in cancer cells but also in immune cells. This Review is composed of 4 different parts, with the impact of alternative splicing in cancer immunotherapy, the role of alternative splicing in immune modulation, the involvement of alternative splicing in cancer cells and finally, the cause of deregulation of alternative splicing in cancer.Splicing is a phenomenon enabling the excision of introns from pre-mRNA to give rise to mature mRNA. All the 20,000 genes of the human genome are concerned by this mechanism. Nevertheless, it is estimated that the proteome is composed of more than 100,000 proteins. How to go from 20,000 genes to more than 100,000 proteins? Alternative splicing (AS) is in charge of this diversity of proteins. AS which is found in most of the cells of an organism, participates in normal cells and in particular in immune cells, in the regulation of cellular behavior. In cancer, AS is highly dysregulated and involved in almost all of the hallmarks that characterize tumor cells. In view of the close link that exists between tumors and the immune system, we present in this review the literature relating to alternative splicing and immunotherapy. We also provide a global but not exhaustive view of AS in the immune system and tumor cells linked to the events that can lead to AS dysregulation in tumors.

Asymmetrical DNA engineering of cell membrane via membrane fusion

Membrane molecules perform a variety of functions that vital to the survival of organisms. Hence, engineer cell membrane with various biomolecules is a promising means to regulate a variety of bioprocess. Because of high programable nature of nucleic acid, engineering of cell plasma membrane using functional DNA has received considerable attention for temporally regulation of cellular behaviors. In addition to temporal regulation, spatial control of functional DNA modifications on cell membranes is equally important to precisely simulate molecules of interest. Particularly, modification of DNA at the intercellular side of membrane is more challenging due to the difficulties of transport negatively charged DNA across cell membrane barrier. Now, writing in Angew . Chem. Int., Ed., Lin and coworkers introduced a membrane fusion-based transport (LiFT) strategy to enable extra- and intra-cellular modification cell membrane with DNA engineering.

Tumor Suppressive Circular RNA-102450: Development of a Novel Diagnostic Procedure for Lymph Node Metastasis from Oral Cancer.

Simple SummaryCircular RNAs (circRNAs) consist of covalently closed structures without a free 3′ poly(A) tail or 5′ cap. Due to their loop structures, circRNAs are largely stable and resistant to RNA degradation by endonucleases. Therefore, they have much longer circulatory half-lives compared with linear RNAs, such as microRNA and long non-coding RNA. CircRNAs have multiple microRNA binding sites, and circRNA binding leads to regulation of the expression of those miRNAs and their target genes by acting as a competing endogenous RNA. However, the underlying molecular mechanisms and clinical correlation between circRNAs and tumor progression are not well understood. In the present study, we identified circRNA-102450 as a novel tumor suppressor and potential biomarker of regional lymph node metastasis from OSCC using liquid biopsy-based droplet digital PCR, a highly sensitive method for absolute quantification.Circular RNAs (circRNAs), which form as covalently closed loop structures, have several biological functions such as regulation of cellular behavior by adsorbing microRNAs. However, there is limited information of circRNAs in oral squamous cell carcinoma (OSCC). Here, we aimed to elucidate the roles of aberrantly expressed circRNAs in OSCC. CircRNA microarray showed that circRNA-102450 was down-regulated in OSCC cells. Clinical validation of circRNA-102450 was performed using highly sensitive droplet digital PCR in preoperative liquid biopsy samples from 30 OSCC patients. Interestingly, none of 16 studied patients with high circRNA-102450 had regional lymph node metastasis (RLNM), whereas 4 of 14 studied patients (28.5%) with low expression had pathologically proven RLNM. Overexpressed circRNA-102450 significantly inhibited the tumor metastatic properties of cell proliferation, migration, and invasion. Furthermore, circRNA-102450 directly bound to, and consequently down-regulated, miR-1178 in OSCC cells. Taken together, circRNA-102450 has a tumor suppressive effect via the circRNA-102450/miR-1178 axis and may be a novel potential marker of RLNM in OSCC patients.

Reversible dynamic mechanics of hydrogels for regulation of cellular behavior

The mechanical properties of the native extracellular matrix play a key role in regulating cell behavior during developmental, healing and homeostatic processes. Since these properties change over time, it may be valuable to have the capacity to dynamically vary the mechanical properties of engineered hydrogels used in tissue engineering strategies to better mimic the dynamic mechanical behavior of native extracellular matrix. However, in situ repeatedly reversible dynamic tuning of hydrogel mechanics is still limited. In this study, we have engineered a hydrogel system with reversible dynamic mechanics using a dual-crosslinkable alginate hydrogel. The effect of reversible mechanical signals on encapsulated stem cells in dynamically tunable hydrogels has been demonstrated. In situ stiffening of hydrogels decreases cell spreading and proliferation, and subsequent softening of hydrogels gives way to an increase in cell spreading and proliferation. The hydrogel stiffening and softening, and resulting cellular responses are repeatedly reversible. This hydrogel system provides a promising platform for investigating the effect of repeatedly reversible changes in extracellular matrix mechanics on cell behaviors. Statement of significanceSince the mechanical properties of native extracellular matrix (ECM) change over time during development, healing and homeostatic processes, it may be valuable to have the capacity to dynamically control the mechanics of biomaterials used in tissue engineering and regenerative medicine applications to better mimic this behavior. Unlike previously reported biomaterials whose mechanical properties can be changed by the user only a limited number of times, this system provides the capacity to induce unlimited alterations to the mechanical properties of an engineered ECM for 3D cell culture. This study presents a strategy for on-demand dynamic and reversible control of materials’ mechanics by single and dual-crosslinking mechanisms using oxidized and methacrylated alginates. By demonstrating direct changes in encapsulated human mesenchymal stem cell morphology, proliferation and chondrogenic differentiation in response to multiple different dynamic changes in hydrogel mechanics, we have established a repeatedly reversible 3D cellular mechanosensing system. This system provides a powerful platform tool with a wide range of stiffness tunability to investigate the role of dynamic mechanics on cellular mechanosensing and behavioral responses.

Laminin-411 and -511 Modulate the Proliferation, Adhesion, and Morphology of Gastric Cancer Cells.

Laminins (Ln), a type of extracellular matrix glycoprotein, are key regulators of cellular behavior. Recent work revealed that in various tumor cell lines, laminin isoforms influence specific responses, such as cell anchorage, survival, proliferation, migration, organization, and specialization. The contribution of laminin isoforms to the function of gastric cancer cells, however, remain unclear. Here, we revealed that in gastric cancer, laminin isoforms Ln411, Ln421, Ln511, and Ln521 promote cellular proliferation; Ln511 and Ln521 increase cell cytoplasmic volume; Ln511 hampers invadopodia formation in some cells, Ln511 enables prompt adhesion of cells to plates, and Ln411 and Ln511 do not alter the gastric cancer stem cell markers CD44 and Lgr5. These results indicate that Ln411 and Ln511 dynamically modulate the proliferation, adhesion, and morphology of gastric cancer cells in different ways that are independent of stem cell properties. In particular, Ln511 showed a high affinity for gastric cancer cells. Our observations broaden the possible options for controlling cancer cell progression and metastasis by modulating laminin-integrin interactions.

Activation of Aptamers with Gain of Function by Small-Molecule-Clipping of Intramolecular Motifs.

Click reactions have the advantages of high reactivity, excellent orthogonality, and synthetic accessibility. Inspired by click reactions, we propose the concept of "clipped aptamers", whose binding affinity is regulated by the "clip"-like specific interaction between a synthetic DNA-mismatch-binding small molecule (molecular glue, Z-NCTS) and the preset CGG/CGG sequences in nucleic acid sequences. In this study, we investigated a Z-NCTS-mediated de novo selection of clipped aptamers against epithelial cell adhesion molecule. The generated clipped aptamers can achieve the efficient transition from a binding-inactive state to an active state by clipping of Z-NCTS with two CGG sites, which otherwise would not hybridize. The experimental and simulation results showed that the clipped aptamer had ideal binding thermodynamics and the ability to regulate cellular adhesion. Because of this superior activated mechanism and structural diversity, clipped aptamers hold great potential in biosensing, imaging, conditional gene- and cellular behavior-regulation, and drug delivery.

Systems Pharmacology Study of the Anticervical Cancer Mechanisms of FDY003

Increasing data support that herbal medicines are beneficial in the treatment of cervical cancer; however, their mechanisms of action remain to be elucidated. In the current study, we used a systems pharmacology approach to explore the pharmacological mechanisms of FDY003, an anticancer herbal formula comprising Lonicera japonica Thunberg, Artemisia capillaris Thunberg, and Cordyceps militaris (Linn.) Link, in the treatment of cervical cancer. Through the pharmacokinetic assessment of absorption-distribution-metabolism-excretion characteristics, we found 18 active compounds that might interact with 106 cervical cancer-related targets responsible for the pharmacological effects. FDY003 targets were significantly associated with gene ontology terms related to the regulation of cellular behaviors, including cell proliferation, cell cycle processes, cell migration, cell apoptosis, cell death, and angiogenesis. The therapeutic targets of the herbal drug were further enriched in various oncogenic pathways that are implicated in the tumorigenesis and progression of cervical cancer, including the phosphatidylinositol 3-kinase, mitogen-activated protein kinase, focal adhesion, human papillomavirus infection, and tumor necrosis factor signaling pathways. Our study provides a systematic approach to explore the anticancer properties of herbal medicines against cervical cancer.

DNA-Based Reprogramming Strategy of Receptor-Mediated Cellular Behaviors: From Genetic Encoding to Nongenetic Engineering.

The precise regulation of fundamental cellular behaviors, such as cell adhesion, proliferation, migration, differentiation, and cell-cell interactions, is essential in multicellular organisms. The regulation of cellular behaviors largely relies on the receptor-mediated sensing and transduction of the extracellular ligands. The precise manipulation of ligand-induced receptor activation via molecular engineering approaches will offer fundamental insight into physiological processes, facilitating the rational design of cell-based therapeutics to treat diseases. DNA is a highly attractive biomolecule as it works through either genetically encoding the functional protein or rationally fabricating functional nanomaterials. Recently accumulated research in functional nucleic acids and DNA nanotechnology makes it possible to construct dynamic and complex nanodevices to modulate receptor-mediated signaling and cellular behaviors. This review summarizes the recent advances in state-of-art of DNA technologies in engineering the interaction between ligand and cell-surface receptor, mainly highlighting the design principles and the emerging applications of DNA-based nongenetic engineering strategies for receptor-mediated cellular behaviors. We expect that significant advancements in DNA-based engineering strategies will enable the emergence of applications in cell-based therapy in regenerative medicine and cancer immunotherapy.

Mesoporous polydopamine with built-in plasmonic core: Traceable and NIR triggered delivery of functional proteins

Functional proteins are essential for the regulation of cellular behaviors and have found growing therapeutic uses. However, low bioavailability of active proteins to their intracellular targets has been a long-standing challenge to achieve their full potential for cell reprogramming and disease treatment. Here we report mesoporous polydopamine (mPDA) with a built-in plasmonic nanoparticle core as a multifunctional protein delivery system. The mPDA with a unique combination of large surface area, metal-chelating property, and broad-spectrum photothermal transduction allows efficient loading and near-infrared light-triggered release of functional proteins, while the plasmonic core serves as a photostable tracer and fluorescence quencher, collectively leading to real-time monitoring and active cytosolic release of model proteins. In particular, controlled delivery of cytotoxic ribonuclease A has shown excellent performance in invivo cancer therapy. The possibility of coating mPDA on a broad range of functional cores, thanks to its universal adhesion, provides opportunities for developing tailored delivery carriers of biologics to overcome intrinsic biological barriers.