Mechanical release of homogenous proteins from supramolecular gels.

A long-standing challenge is how to formulate proteins and vaccines to retain function during storage and transport and to remove the burdens of cold-chain management. Any solution must be practical to use, with the protein being released or applied using clinically relevant triggers. Advanced biologic therapies are distributed cold, using substantial energy, limiting equitable distribution in low-resource countries and placing responsibility on the user for correct storage and handling. Cold-chain management is the best solution at present for protein transport but requires substantial infrastructure and energy. For example, in research laboratories, a single freezer at -80 °C consumes as much energy per day as a small household1. Of biological (protein or cell) therapies and all vaccines, 75% require cold-chain management; the cost of cold-chain management in clinical trials has increased by about 20% since 2015, reflecting this complexity. Bespoke formulations and excipients are now required, with trehalose2, sucrose or polymers3 widely used, which stabilize proteins by replacing surface water molecules and thereby make denaturation thermodynamically less likely; this has enabled both freeze-dried proteins and frozen proteins. For example, the human papilloma virus vaccine requires aluminium salt adjuvants to function, but these render it unstable against freeze-thaw4, leading to a very complex and expensive supply chain. Other ideas involve ensilication5 and chemical modification of proteins6. In short, protein stabilization is a challenge with no universal solution7,8. Here we designed a stiff hydrogel that stabilizes proteins against thermal denaturation even at 50 °C, and that can, unlike present technologies, deliver pure, excipient-free protein by mechanically releasing it from a syringe. Macromolecules can be loaded at up to 10 wt% without affecting the mechanism of release. This unique stabilization and excipient-free release synergy offers a practical, scalable and versatile solution to enable the low-cost, cold-chain-free and equitable delivery of therapies worldwide.

A bioorthogonal system reveals antitumour immune function of pyroptosis.

Bioorthogonal chemistry capable of operating in live animals is needed to investigate biological processes such as cell death and immunity. Recent studies have identified a gasdermin family of pore-forming proteins that executes inflammasome-dependent and -independent pyroptosis1-5. Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown. Here we establish a bioorthogonal chemical system, in which a cancer-imaging probe phenylalanine trifluoroborate (Phe-BF3) that can enter cells desilylates and 'cleaves' a designed linker that contains a silyl ether. This system enabled the controlled release of a drug from an antibody-drug conjugate in mice. When combined with nanoparticle-mediated delivery, desilylation catalysed by Phe-BF3 could release a client protein-including an active gasdermin-from a nanoparticle conjugate, selectively into tumour cells in mice. We applied this bioorthogonal system to gasdermin, which revealed that pyroptosis of less than 15% of tumour cells was sufficient to clear the entire 4T1 mammary tumour graft. The tumour regression was absent in immune-deficient mice or upon T cell depletion, and was correlated with augmented antitumour immune responses. The injection of a reduced, ineffective dose of nanoparticle-conjugated gasdermin along with Phe-BF3 sensitized 4T1 tumours to anti-PD1 therapy. Our bioorthogonal system based on Phe-BF3 desilylation is therefore a powerful tool for chemical biology; our application of this system suggests that pyroptosis-induced inflammation triggers robust antitumour immunity and can synergize with checkpoint blockade.

The strawberry-derived permeation enhancer pelargonidin enables oral protein delivery.

Although patients generally prefer oral drug delivery to injections, low permeability of the gastrointestinal tract makes this method impossible for most biomacromolecules. One potential solution is codelivery of macromolecules, including therapeutic proteins or nucleic acids, with intestinal permeation enhancers; however, enhancer use has been limited clinically by modest efficacy and toxicity concerns surrounding long-term administration. Here, we hypothesized that plant-based foods, which are well tolerated by the gastrointestinal tract, may contain compounds that enable oral macromolecular absorption without causing adverse effects. Upon testing more than 100 fruits, vegetables, and herbs, we identified strawberry and its red pigment, pelargonidin, as potent, well-tolerated enhancers of intestinal permeability. In mice, an oral capsule formulation comprising pelargonidin and a 1 U/kg dose of insulin reduced blood glucose levels for over 4 h, with bioactivity exceeding 100% relative to subcutaneous injection. Effects were reversible within 2 h and associated with actin and tight junction rearrangement. Furthermore, daily dosing of mice with pelargonidin for 1 mo resulted in no detectable side effects, including weight loss, tissue damage, or inflammatory responses. These data suggest that pelargonidin is an exceptionally effective enhancer of oral protein uptake that may be safe for routine pharmaceutical use.

Indocyanine Green-Loaded Adhesive Proteinic Nanoparticles for Effective Locoregional and Prolonged Photothermal Anticancer Therapy.

Conventional anticancer therapies, including surgical resection, radiation, and chemotherapy, are the primary modalities for treating various forms of cancer. However, these treatments often bring significant side effects and risk of recurrence, underscoring the need for more targeted and less invasive therapeutic options. To address this challenge, we developed an adhesive nanoparticle (NP)-based effective anticancer photothermal therapy (PTT) system using bioengineered mussel adhesion protein (MAP). The unique underwater tissue adhesive properties of MAP NPs enabled targeted delivery and prolonged retention at the tumor site, thereby improving therapeutic efficacy. Our innovative indocyanine green (ICG)-loaded MAP NPs (MAP@ICG NPs) demonstrated strong photothermal capability and stability, and potent anticancer activity in vitro. In vivo intratumor injection of the MAP@ICG NPs showed remarkable anticancer PTT effects, effectively reducing tumor growth with minimal damage to surrounding tissues. The development and utilization of this adhesive proteinic NP-based PTT system represent a significant advancement in cancer therapy, offering a promising alternative that combines the precision of NP delivery with effective therapeutic efficacy.

Engineered Protein Scaffolds as Next-Generation Therapeutics.

The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology. Early examples were the Affibody, Monobody (Adnectin), and Anticalin proteins, which were derived from fragments of streptococcal protein A, from the tenth type III domain of human fibronectin, and from natural lipocalin proteins, respectively. Since then, this concept has expanded considerably, including many other protein templates. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. However, despite strong interest from basic science, only a handful of those protein scaffolds have undergone biopharmaceutical development up to the clinical stage. This includes the abovementioned pioneering examples as well as designed ankyrin repeat proteins (DARPins). Here we review the current state and clinical validation of these next-generation therapeutics.

Emergence in protein derived nanomedicine as anticancer therapeutics: More than a tour de force.

Cancer has thwarted as a major health problem affecting the global population. With an alarming increase in the patient population suffering from diverse varieties of cancers, the global demographic data predicts sharp escalation in the number of cancer patients. This can be expected to reach 420 million cases by 2025. Among the diverse types of cancers, the most frequently diagnosed cancers are the breast, colorectal, prostate and lung cancer. From years, conventional treatment approaches like surgery, chemotherapy and radiation therapy have been practiced. In the past few years, increasing research on molecular level diagnosis and treatment of cancers have significantly changed the realm of cancer treatment. Lately, uses of advanced chemotherapy and immunotherapy like treatments have gained significant progress in the cancer therapy, but these approaches have several limitations on their safety and toxicity. This has generated lot of momentum for the evolution of new drug delivery approaches for the effective delivery of anticancer therapeutics, which may improve the pharmacokinetic and pharmacodynamic effect of the drugs along with significant reduction in the side effects. In this regard, the protein-based nano-medicines have gained wider attention in the management of cancer. Proteins are organic macromolecules essential, for life and have quite well explored in developing the nano-carriers. Furthermore, it provides passive or active tumour cell targeted delivery, by using protein based nanovesicles or virus like structures, antibody drug conjugates, viral particles, etc. Moreover, by utilizing various formulation strategies, both the animal and plant derived proteins can be converted to produce self-assembled virus like nano-metric structures with high efficiency in targeting the metastatic cancer cells. Therefore, the present review extensively discusses the applications of protein-based nano-medicine with special emphasis on intracellular delivery/drug targeting ability for anticancer drugs.

Novel therapeutic interventions in cancer treatment using protein and peptide-based targeted smart systems.

Cancer, being the most prevalent and resistant disease afflicting any gender, age or social status, is the ultimate challenge for the scientific community. The new generation therapeutics for cancer management has shifted the approach to personalized/precision medicine, making use of patient- and tumor-specific markers for specifying the targeted therapies for each patient. Peptides targeting these cancer-specific signatures hold enormous potential for cancer therapy and diagnosis. The rapid advancements in the combinatorial peptide libraries served as an impetus to the development of multifunctional peptide-based materials for targeted cancer therapy. The present review outlines benefits and shortcomings of peptides as cancer therapeutics and the potential of peptide modified nanomedicines for targeted delivery of anticancer agents.

The human IL-15 superagonist N-803 promotes migration of virus-specific CD8+ T and NK cells to B cell follicles but does not reverse latency in ART-suppressed, SHIV-infected macaques.

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.

A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties.

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

Intralesional Infiltrations of Arteriosclerotic Tissue Cells-Free Filtrate Reproduce Vascular Pathology in Healthy Recipient Rats.

Lower-extremity arterial disease is a major health problem with increasing prevalence, often leading to non-traumatic amputation, disability and mortality. The molecular mechanisms underpinning abnormal vascular wall remodeling are not fully understood. We hypothesized on the existence of a vascular tissue memory that may be transmitted through soluble signaling messengers, transferred from humans to healthy recipient animals, and consequently drive the recapitulation of arterial wall thickening and other vascular pathologies. We examined the effects of the intralesional infiltration for 6 days of arteriosclerotic popliteal artery-derived homogenates (100 µg of protein) into rats' full-thickness wounds granulation tissue. Animals infiltrated with normal saline solution or healthy brachial arterial tissue homogenate obtained from traumatic amputation served as controls. The significant thickening of arteriolar walls was the constant outcome in two independent experiments for animals receiving arteriosclerotic tissue homogenates. This material induced other vascular morphological changes including an endothelial cell phenotypic reprogramming that mirrored the donor's vascular histopathology. The immunohistochemical expression pattern of relevant vascular markers appeared to match between the human tissue and the corresponding recipient rats. These changes occurred within days of administration, and with no cross-species limitation. The identification of these "vascular disease drivers" may pave novel research avenues for atherosclerosis pathobiology.

Rapid protein delivery to living cells for biomolecular investigation.

The lack of a simple, fast and efficient method for protein delivery is limiting the widespread application of in-cell experiments, which are crucial for understanding the cellular function. We present here an innovative strategy to deliver proteins into both prokaryotic and eukaryotic cells, exploiting thermal vesiculation. This method allows to internalize substantial amounts of proteins, with different molecular weight and conformation, without compromising the structural properties and cell viability. Characterizing proteins in a physiological environment is essential as the environment can dramatically affect the conformation and dynamics of biomolecules as shown by in-cell EPR spectra vs those acquired in buffer solution. Considering its versatility, this method opens the possibility to scientists to study proteins directly in living cells through a wide range of techniques.

Analysis of daily energy, protein, fat, and carbohydrate intake in citrin-deficient patients: Towards prevention of adult-onset type II citrullinemia.

Patients with citrin deficiency during the adaptation/compensation period exhibit diverse clinical features and have characteristic diet of high protein, high fat, and low carbohydrate. Japanese cuisine typically contains high carbohydrate but evaluation of diet of citrin-deficient patients in 2008 showed a low energy intake and a protein:fat:carbohydrate (PFC) ratio of 19:44:37, which indicates low carbohydrate consumption rate. These findings prompted the need for diet intervention to prevent the adult onset of type II citrullinemia (CTLN2). Since the publication of the report about 10 years ago, patients are generally advised to eat what they wish under active dietary consultation and intervention. In this study, citrin-deficient patients and control subjects living in the same household provided answers to a questionnaire, filled-up a maximum 6-day food diary, and supplied physical data and information on medications if any. To study the effects of the current diet, the survey collected data from 62 patients and 45 controls comparing daily intakes of energy, protein, fat, and carbohydrate. Food analysis showed that patient's energy intake was 115% compared to the Japanese standard. The confidence interval of the PFC ratio of patients was 20-22:47-51:28-32, indicating higher protein, higher fat and lower carbohydrate relative to previous reports. The mean PFC ratio of female patients (22:53:25) was significantly different from that of male patients (20:46:34), which may explain the lower frequency of CTLN2 in females. Comparison of the present data to those published 10 years ago, energy, protein, and fat intakes were significantly higher but the amount of carbohydrate consumption remained the same. Regardless of age, most patients (except for adolescents) consumed 100-200 g/day of carbohydrates, which met the estimated average requirement of 100 g/day for healthy individuals. Finally, patients were generally not overweight and some CTLN2 patients were underweight although their energy intake was higher compared with the control subjects. We speculate that high-energy of a low carbohydrate diet under dietary intervention may help citrin-deficient patients attain normal growth and prevent the onset of CTLN2.

The impact of low muscle function on health-related quality of life in Indonesian women with systemic lupus erythematosus.

OBJECTIVE: There was no study aimed at evaluating the effect of muscle function on SLE patients' quality of life using the Sarcopenia Quality of Life (SarQoL) questionnaire. METHODS: This cross-sectional study recruited 61 women with SLE consecutively, muscle function was measured with Jamar handheld-dynamometer and 6-meter walk test, HRQoL was measured with Sarcopenia Quality of Life (SarQoL) questionnaire. The cut-off point for low muscle strength (<18 kg) and low gait speed (<1.0 m/s) was according to the Asian Working Group on Sarcopenia 2019 criteria. Statistical analysis was conducted with a t-test for mean difference, and linear regression was used to adjust confounders (age, protein intake, physical exercise, and disease activity). RESULTS: The subjects' mean muscle strength was 19.54 kg (6.94), and 44.3% (n = 27) was found to have low muscle strength. The subjects' mean gait speed was 0.77 m/s (0.20), and 90.3% (n = 55) was found to have low gait speed. The difference of total SarQoL score in subjects with normal and low muscle strength was found to be significant; 74.86 (9.48) vs. 65.49 (15.51) (p = 0.009), and still statistically significant after adjustments with age, protein intake, physical exercise level, and disease activity [B 0.56; 95% CI 0.08-1.03; p = 0.022]. The difference of total SarQoL score in subjects with normal and low physical performance was found to be not significant, 70.67 (11.08) vs. 70.72 (13.56) (p = 0.993). CONCLUSION: There was a significant difference in SarQoL's total score in normal compared with low muscle strength groups of Indonesian women with SLE.

Combined Application of Prototype Ultrasound and BSA-Loaded PLGA Particles for Protein Delivery.

PURPOSE: To develop an in vitro culture system for tissue engineering to mimic the in vivo environment and evaluate the applicability of ultrasound and PLGA particle system. METHODS: For tissue engineering, large molecules such as growth factors for cell differentiation should be supplied in a controlled manner into the culture system, and the in vivo microenvironment need to be reproduced in the system for the regulation of cellular function. In this study, portable prototype ultrasound with low intensity was devised and tested for protein release from bovine serum albumin (BSA)-loaded poly(lactic-co-glycolic acid) (PLGA) particles. RESULTS: BSA-loaded PLGA particles were prepared using various types of PLGA reagents and their physicochemical properties were characterized including particle size, shape, or aqueous wetting profiles. The BSA-loaded formulation showed nano-ranged size distribution with optimal physical stability during storage period, and protein release behaviors in a controlled manner. Notably, the application of prototype ultrasound with low intensity influenced protein release patterns in the culture system containing the BSA-loaded PLGA formulation. The results revealed that the portable ultrasound set controlled by the computer could contribute for the protein delivery in the culture medium. CONCLUSIONS: This study suggests that combined application with ultrasound and protein-loaded PLGA encapsulation system could be utilized to improve culture system for tissue engineering or cell regeneration therapy.

Lysosomal escaped protein nanocarriers for nuclear-targeted siRNA delivery.

In the process of drug carrier design, lysosome degradation in cells is often neglected, which makes a considerable number of drugs not play a role. Here, we have constructed a tumor treatment platform (Apn/siRNA/NLS/HA/Apt) with unique lysosomal escape function and excellent cancer treatment effect. Apoferritin (Apn) has attracted more and more attention because of its high uniformity, modifiability, and controllability. Meanwhile, its endogenous nature can avoid the risk of immune response being eliminated. We used aptamer modified iron deficient protein nanocages (Apn) to tightly encapsulate the combination of siRNA and NLS (siRNA/NLS) with influenza virus hemagglutinin (HA peptide). After Apn/siRNA/NLS/HA/Apt was targeted into cells, the acidic environment of lysosome led to the cleavage of Apn nanocages, and the release of siRNA/NLS and HA peptide. HA peptide can destroy lysosome membrane, make siRNA/NLS escape lysosome, and enter the nucleus under the action of NLS, resulting in efficient gene silencing effect. This kind of cancer treatment strategy based on Apn nanocage shows high biocompatibility and unique lysosome escape property, which significantly improves the drug delivery and treatment efficiency. Lysosomal escape protein nanocarriers for nuclear-targeted siRNA delivery.

Feasibility of achieving different protein targets using a hypocaloric high-protein enteral formula in critically ill patients.

BACKGROUND AND AIMS: Combining energy and protein targets during the acute phase of critical illness is challenging. Energy should be provided progressively to reach targets while avoiding overfeeding and ensuring sufficient protein provision. This prospective observational study evaluated the feasibility of achieving protein targets guided by 24-h urinary nitrogen excretion while avoiding overfeeding when administering a high protein-to-energy ratio enteral nutrition (EN) formula. METHODS: Critically ill adult mechanically ventilated patients with an APACHE II score > 15, SOFA > 4 and without gastrointestinal dysfunction received EN with hypocaloric content for 7 days. Protein need was determined by 24-h urinary nitrogen excretion, up to 1.2 g/kg (Group A, N = 10) or up to 1.5 g/kg (Group B, N = 22). Variables assessed included nitrogen intake, excretion, balance; resting energy expenditure (REE); phase angle (PhA); gastrointestinal tolerance of EN. RESULTS: Demographic characteristics of groups were similar. Protein target was achieved using urinary nitrogen excretion measurements. Nitrogen balance worsened in Group A but improved in Group B. Daily protein and calorie intake and balance were significantly increased in Group B compared to Group A. REE was correlated to PhA measurements. Gastric tolerance of EN was good. CONCLUSIONS: Achieving the protein target using urinary nitrogen loss up to 1.5 g/kg/day was feasible in this hypercatabolic population. Reaching a higher protein and calorie target did not induce higher nitrogen excretion and was associated with improved nitrogen balance and a better energy intake without overfeeding. PhA appears to be related to REE and may reflect metabolism level, suggestive of a new phenotype for nutritional status. Trial registration 0795-18-RMC.

PLGA/PLA-Based Long-Acting Injectable Depot Microspheres in Clinical Use: Production and Characterization Overview for Protein/Peptide Delivery.

Over the past few decades, long acting injectable (LAI) depots of polylactide-co-glycolide (PLGA) or polylactic acid (PLA) based microspheres have been developed for controlled drug delivery to reduce dosing frequency and to improve the therapeutic effects. Biopharmaceuticals such as proteins and peptides are encapsulated in the microspheres to increase their bioavailability and provide a long release period (days or months) with constant drug plasma concentration. The biodegradable and biocompatible properties of PLGA/PLA polymers, including but not limited to molecular weight, end group, lactide to glycolide ratio, and minor manufacturing changes, could greatly affect the quality attributes of microsphere formulations such as release profile, size, encapsulation efficiency, and bioactivity of biopharmaceuticals. Besides, the encapsulated proteins/peptides are susceptible to harsh processing conditions associated with microsphere fabrication methods, including exposure to organic solvent, shear stress, and temperature fluctuations. The protein/peptide containing LAI microspheres in clinical use is typically prepared by double emulsion, coacervation, and spray drying techniques. The purpose of this review is to provide an overview of the formulation attributes and conventional manufacturing techniques of LAI microspheres that are currently in clinical use for protein/peptides. Furthermore, the physicochemical characteristics of the microsphere formulations are deliberated.

Increase in Brain Volumes after Implementation of a Nutrition Regimen in Infants Born Extremely Preterm.

OBJECTIVE: To assess the effect of early life nutrition on structural brain development in 2 cohorts of extremely preterm infants, before and after the implementation of a nutrition regimen containing more protein and lipid. STUDY DESIGN: We included 178 infants retrospectively (median gestational age, 26.6 weeks; IQR, 25.9-27.3), of whom 99 received the old nutrition regimen (cohort A, 2011-2013) and 79 the new nutrition regimen (cohort B, 2013-2015). Intake of protein, lipids, and calories was calculated for the first 28 postnatal days. Brain magnetic resonance imaging (MRI) was performed at 30 weeks postmenstrual age (IQR, 30.3-31.4) and term-equivalent age (IQR, 40.9-41.4). Volumes of 42 (left + right) brain structures were calculated. RESULTS: Mean protein and caloric intake in cohort B (3.4 g/kg per day [P < .001] and 109 kcal/kg per day [P = .038]) was higher than in cohort A (2.7 g/kg per day; 104 kcal/kg per day). At 30 weeks, 22 regions were significantly larger in cohort B compared with cohort A, whereas at term-equivalent age, only the caudate nucleus was significantly larger in cohort B compared with cohort A. CONCLUSIONS: An optimized nutrition protocol in the first 28 days of life is associated with temporarily improved early life brain volumes.

Site-Specific Bioconjugation Approaches for Enhanced Delivery of Protein Therapeutics and Protein Drug Carriers.

Proteins have the capacity to treat a multitude of diseases both as therapeutics and as drug carriers due to their complex functional properties, specificity toward binding partners, biocompatibility, and programmability. Despite this, native proteins often require assistance to target diseased tissue due to poor pharmacokinetic properties and membrane impermeability. Functionalizing therapeutic proteins and drug carriers through direct conjugation of delivery moieties can enhance delivery capabilities. Traditionally, this has been accomplished through bioconjugation methods that have little control over the location or orientation of the modification, leading to highly heterogeneous products with varying activity. A multitude of promising site-specific protein conjugation methods have been developed to allow more tailorable display of delivery moieties and thereby enhance protein activity, circulation properties, and targeting specificity. Here, we focus on three particularly promising site-specific bioconjugation techniques for protein delivery: unnatural amino acid incorporation, Sortase-mediated ligation, and SpyCatcher/SpyTag chemistry. In this review, we highlight the promise of site-specific bioconjugation for targeted drug delivery by summarizing impactful examples in literature, considering important design principles when constructing bioconjugates, and discussing our perspectives on future directions.