Targeted Delivery Of Proteins Research Articles

Polypyridiniums with Inherent Autophagy-Inducing Activity for Atherosclerosis Treatment by Intracellularly Co-Delivering Two Antioxidant Enzymes.

Atherosclerosis is a chronic inflammatory disease of the arterial intima and is becoming the leading cause of morbidity and mortality worldwide. There is considerable evidence that defective autophagy and overproduction of reactive oxygen species (ROS) are closely involved in the development and progression of atherosclerosis. Here, a polymer is developed with the inherent autophagy-inducing activity to treat atherosclerosis by co-delivering antioxidant enzymes. The lead material P5c screened from a library of polypyridiniums shows robust efficacy in cytosolic protein delivery, and efficiently delivers superoxide dismutase (SOD) and catalase (CAT) into macrophages to down-regulate intracellular ROS. Moreover, P5c activates autophagy in macrophages and sufficiently inhibits foam cell formation. The P5c nanoparticle loaded with both SOD and CAT is further coated with neutrophil membranes to treat atherosclerosis in an ApoE-/- mice model. The treatment exhibits potent anti-atherosclerosis effect via activating autophagy, decreasing the infiltration of senescent cells in atherosclerotic plaques, regulating the M2 polarization of macrophages, and restoring the structure and function of splenic corpuscles. The polymer offers a multifaceted approach to combat atherosclerosis, addressing both cellular dysfunction and the need for targeted protein delivery within affected cells.

Develop Targeted Protein Drug Carriers through a High-Throughput Screening Platform and Rational Design.

Protein-based drugs offer advantages, such as high specificity, low toxicity, and minimal side effects compared to small molecule drugs. However, delivery of proteins to target tissues or cells remains challenging due to the instability, diverse structures, charges, and molecular weights of proteins. Polymers have emerged as a leading choice for designing effective protein delivery systems, but identifying a suitable polymer for a given protein is complicated by the complexity of both proteins and polymers. To address this challenge, a fluorescence-based high-throughput screening platform called ProMatch to efficiently collect data on protein-polymer interactions, followed by in vivo and in vitro experiments with rational design is developed. Using this approach to streamline polymer selection for targeted protein delivery, candidate polymers from commercially available options are identified and a polyhexamethylene biguanide (PHMB)-based system for delivering proteins to white adipose tissue as a treatment for obesity is developed. A branched polyethylenimine (bPEI)-based system for neuron-specific protein delivery to stimulate optic nerve regeneration is also developed. The high-throughput screening methodology expedites identification of promising polymer candidates for tissue-specific protein delivery systems, thereby providing a platform to develop innovative protein-based therapeutics.

Porphysome Engineered With Specific Protein Binding Sites as a Multimodal Theranostic Nanocarrier for Targeted Protein Delivery.

The immobilization of proteins on the surface of carriers is challenging due to the loss of protein structure and function in this process. Here, we report the development of the protein immobilization on the surface of the metallated-porphyrin complex in the porphysome nanocarrier. The conjugated Ni-porphyrin to fatty acid (as a tail) has been synthesized and independently placed at the depth of the bilayer center of Dipalmitoylphosphatidylcholine (DPPC) in which the Ni-porphyrin was at the polar region of the membrane and is thus superficial. This porphysome (DPPC: Ni-porphyrin, 4 : 1 mole ratio) was formed by supramolecular self-assembly with a diameter of 173±7 nm and zeta potential -8.5±3.4 mv, which exhibited no significant toxicity at the experimental concentrations and acceptable cellular uptake on MCF-7 cells. The physicochemical properties and specific protein binding sites of the firefly luciferase as a model protein into the porphysome (1 : 2 mole ratio) show the conjugation efficiency about 80 % and the conformation of protein was completely maintained. Furthermore, bioluminescence assay and SDS-PAGE confirmed the preservation of protein function. The stabilized platform of porphyrin-lipid structure can potentially improve the efficacy of protein functionality for a particular display, shifting porphysomes from a simple carrier to a therapeutic agent.

Aptamer and phosphorothioated DNA engineered liposomes as a targeted intracellular protein delivery system

Targeted delivery of proteins into desired cell groups is crucial for disease therapy and cellular functionalization. However, current delivery methods face severe side effects and off-target problems, making it difficult to achieve cell-targeted protein delivery. Herein, we developed a user-friendly membrane fusion liposome for cancer cell-targeted protein delivery. Phosphorothioated DNA-mediated membrane fusion was employed as an efficient transmembrane delivery approach. The phosphorothioated DNA was capped with the AS1411 aptamer, which specifically recognizes nucleolar proteins on the cancer cell membrane, enabling a controllable delivery of proteins into targeted cancer cells. This delivery system exhibited commendable biocompatibility and targeted delivery ability, thereby realizing highly effective cancer cell inhibition in vitro. The in vivo results further suggested that the membrane protein-responsive membrane fusion delivery system offers a new avenue for highly biocompatible and targeted protein delivery.

Targeted protein delivery based on stimuli‐triggered nanomedicine

AbstractProtein‐based drugs have shown unique advantages to treat various diseases in recent years. However, most protein therapeutics in clinical use are limited to extracellular targets with low delivery efficiency. To realize targeted protein delivery, a series of stimuli‐triggered nanoparticle formulations have been developed to improve delivery efficiency and reduce off‐target release. These smart nanoparticles are designed to release cargo proteins in response to either internal or external stimuli at pathological tissues. In this way, varieties of protein‐based drugs including antibodies, enzymes, and pro‐apoptotic proteins can be effectively delivered to desired sites for the treatment of cancer, inflammation, metabolic diseases, and so on with minimal side effects. In this review, recent advances in the design of stimuli‐triggered nanomedicine for targeted protein delivery in different biomedical applications will be discussed. A deeper understanding of these emerging strategies helps develop more efficient protein delivery systems for clinical use in the future.

Construction and Functionalization of a Clathrin Assembly for a Targeted Protein Delivery (Small 8/2023)

Targeted Protein Deliveries In article number 2204620, Hak-Sung Kim and co-workers develop a new type of protein self-assembly using human clathrin for a targeted protein delivery. A redesigned clathrin triskelion is dual-functionalized with functional biomolecules. The resulting clathrin assembly exhibits a significantly high avidity and an efficient delivery of a protein cargo into tumor cells.

Construction and Functionalization of a Clathrin Assembly for a Targeted Protein Delivery.

Protein assemblies have drawn much attention as platforms for biomedical applications, including gene/drug delivery and vaccine, due to biocompatibility and functional diversity. Here, the construction and functionalization of a protein assembly composed of human clathrin heavy chain and light chain for a targeted protein delivery, is presented. The clathrin heavy and light chains are redesigned and associated with each other, and the resulting triskelion unit further self-assembled into a clathrin assembly with the size of about 28nm in diameter. The clathrin assembly is dual-functionalized with a protein cargo and a targeting moiety using two different orthogonal protein-ligand pairs through one-pot reaction. The functionalized clathrin assembly exhibits about a 900-fold decreased KD value for a cell-surface target due to avidity compared to a native targeting moiety. The utility of the clathrin assembly is demonstrated by an efficient delivery of a protein cargo into tumor cells in a target-specific manner, resulting in a strong cytotoxic effect. The present approach can be used in the creation of protein assemblies with multimodality.

Transferrin Aptamers Increase the In Vivo Blood-Brain Barrier Targeting of Protein Spherical Nucleic Acids.

The systemic delivery of exogenous proteins to cells within the brain and central nervous system (CNS) is challenging due to the selective impermeability of the blood-brain barrier (BBB). Herein, we hypothesized that protein delivery to the brain could be improved via functionalization with DNA aptamers designed to bind transferrin (TfR) receptors present on the endothelial cells that line the BBB. Using β-galactosidase (β-Gal) as a model protein, we synthesized protein spherical nucleic acids (ProSNAs) comprised of β-Gal decorated with TfR aptamers (Transferrin-ProSNAs). The TfR aptamer motif significantly increases the accumulation of β-Gal in brain tissue in vivo following intravenous injection over both the native protein and ProSNAs containing nontargeting DNA sequences. Furthermore, the widespread distribution of β-Gal throughout the brain is only observed for Transferrin-ProSNAs. Together, this work shows that the SNA architecture can be used to selectively deliver protein cargo to the brain and CNS if the appropriate aptamer sequence is employed as the DNA shell. Moreover, this highlights the importance of DNA sequence design and provides a potential new avenue for designing highly targeted protein delivery systems by combining the power of DNA aptamers together with the SNA platform.

Incorporation of Endosomolytic Peptides with Varying Disruption Mechanisms into EGFR-Targeted Protein Conjugates: The Effect on Intracellular Protein Delivery and EGFR Specificity in Breast Cancer Cells.

Intracellular delivery of protein therapeutics remains a significant challenge limiting the majority of clinically available protein drugs to extracellular targets. Strategies to deliver proteins to subcellular compartments have traditionally relied on cell-penetrating peptides, which can drive enhanced internalization but exhibit unreliable activity and are rarely able to target specific cells, leading to off-target effects. Moreover, few design rules exist regarding the relative efficacy of various endosomal escape strategies in proteins. Accordingly, we developed a simple fusion modification approach to incorporate endosomolytic peptides onto epidermal growth factor receptor (EGFR)-targeted protein conjugates and performed a systematic comparison of the endosomal escape efficacy, mechanism of action, and capacity to maintain EGFR-targeting specificity of conjugates modified with four different endosomolytic sequences of varying modes of action (Aurein 1.2, GALA, HA2, and L17E). Use of the recently developed Gal8-YFP assay indicated that the fusion of each endosomolytic peptide led to enhanced endosomal disruption. Additionally, the incorporation of each endosomolytic peptide increased the half-life of the internalized protein and lowered lysosomal colocalization, further supporting the membrane-disruptive capacity. Despite this, only EGFR-targeted conjugates modified with Aurein 1.2 or GALA maintained EGFR specificity. These results thus demonstrated that the choice of endosomal escape moiety can substantially affect targeting capability, cytotoxicity, and bioactivity and provided important new insights into endosomolytic peptide selection for the design of targeted protein delivery systems.

Oligosaccharide-lactoferrin shell-crosslinked particles for selective targeting of proteins to probiotic bacteria in the colon

Consumption of prebiotics seems to be a more effective approach for improving health compared to consumption of probiotic bacteria. However, these beneficial microorganisms require proteins to proliferate, while most consumed protein does not reach the gut. Still, no protein-containing prebiotics are commercially-available. Probiotic bacteria express sugar-binding-proteins (SBP) on their cell-membrane that mediate uptake of prebiotic oligosaccharides (OS). We have recently shown that Galactooligosaccharide-Lactoferrin hydrolysate (GOS-LFH) Maillard conjugates can serve as a vehicle for targeted delivery of protein to a model probiotic bacterium (Lactobacillus casei), boosting its growth rate. Herein, we aimed to advance this targeted protein delivery system by preparing three different compositions of prebiotic OS-LFH (2′-Fucosyllactose-LFH (2′-FL-LFH), Fructooligosaccharide-LFH (FOS-LFH), and Xylooligosaccharide-LFH (XOS-LFH)) self-assembled micellar particles, and characterizing their structure, physicochemical properties, and in-vitro digestibility. The OS shell of the particles is the molecular-recognition ligand for selective targeting to probiotic's SBP, and a shield against premature digestion of the delivered protein. We have demonstrated that these OS-LFH conjugates self-assembled into 10–25 nm particles, exhibiting good colloidal stability and lower susceptibility to proteolysis compared to the native lactoferrin (LF). Shell-crosslinking significantly reduced in-vitro gastrointestinal digestibility of the protein core. The proteolysis of the crosslinked 2′-FL-LFH, FOS-LFH, XOS-LFH progressed to only 55%, 57%, and 68% respectively, compared to native LF. Collectively, our findings suggest that these OS-LFH particles embody a promising strategy for high endurance and selective targeted delivery of protein to gut probiotics, which may enhance their competitive edge over non-beneficial/harmful colon bacteria, thereby promoting their potential to improve health.

Light-Controlled Release of Therapeutic Proteins from Red Blood Cells

Protein therapeutics are a powerful class of drugs known for their selectivity and potency. However, the potential efficacy of these therapeutics is commonly offset by short circulatory half-lives and undesired action at otherwise healthy tissue. We describe herein a targeted protein delivery system that employs engineered red blood cells (RBCs) as carriers and light as the external trigger that promotes hemolysis and drug release. RBCs internally loaded with therapeutic proteins are readily surface modified with a dormant hemolytic peptide. The latter is activated via easily assigned wavelengths that extend into the optical window of tissue. We have demonstrated that photorelease transpires with spatiotemporal control and that the liberated proteins display the anticipated biological effects in vitro. Furthermore, we have confirmed targeted delivery of a clot-inducing enzyme in a mouse model. Finally, we anticipate that this strategy is not limited to RBC carriers but also should be applicable to nano- and microtransporters comprised of bilayer lipid membranes.

A pH-activated charge convertible quantum dot as a novel nanocarrier for targeted protein delivery and real-time cancer cell imaging.

The rapid developments of nanocarriers based on quantum dots (QDs) have been confirmed to show substantial promise for drug delivery and bioimaging. However, optimal QDs-based nanocarriers still need to have their controlled behavior in vitro and in vivo and decrease heavy metal-associated cytotoxicity. Herein, a pH-activated charge convertible QD-based nanocarrier was fabricated by capping multifunctional polypeptide ligands (mPEG-block-poly(ethylenediamine-dihydrolipoic acid-2,3-dimethylmaleic anhydride)-L-glutamate, PEG-P(ED-DLA-DMA)LG) onto the surface of core/multishell CdSe@ZnS/ZnS QD by means of a ligand exchange strategy, followed by uploading of cytochrome C (CC) (CC-loaded QD-PEG-P(ED-DLA-DMA)LG) via electrostatic interactions, in which QDs that were water-soluble and protein-loading were perfectly integrated. That is, the CC-loaded QD-PEG-P(ED-DLA-DMA)LG inherited excellent fluorescence properties from CdSe@ZnS/ZnS QD for real-time imaging, as well as tumor-microenvironment sensitivities from PEG-P(ED-DLA-DMA)LG for enhanced cellular uptake and CC release. Experimental results verified that the QD-PEG-P(ED-DLA-DMA)LG showed enhanced internalization, rapid endo/lysosomal escape, and supplied legible real-time imaging for lung carcinoma cells. Furthermore, pH-triggered charge-convertible ability enabled the QD-PEG-P(ED-DLA-DMA)LG-CC to effectively kill cancer cells better than did the control groups. Hence, constructing smart nanocomposites by facile ligand-exchange strategy is beneficial to QD-based nanocarrier for tumor-targeting cancer therapy.

Anthrax Protective Antigen Retargeted with Single-Chain Variable Fragments Delivers Enzymes to Pancreatic Cancer Cells.

The nontoxic, anthrax protective antigen/lethal factor N-terminal domain (PA/LFN ) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LFN to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single-chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer-relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA-scFv fusions enable cell-specific delivery of diphtheria toxin catalytic domain and Ras/Rap1-specific endopeptidase to pancreatic cancer cells. Importantly, mPA-scFv fusion-based treatments display potent cell-specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells.

Targeted Protein Delivery: Supramolecular Toxin Complexes for Targeted Pharmacological Modulation of Polymorphonuclear Leukocyte Functions (Adv. Healthcare Mater. 17/2019)

Targeted Protein Delivery: Supramolecular Toxin Complexes for Targeted Pharmacological Modulation of Polymorphonuclear Leukocyte Functions (Adv. Healthcare Mater. 17/2019)

Modulated Protein Delivery to Engineer Tissue Repair.

Achieving targeted protein delivery to injured tissues is a core focus of the field of tissue engineering and has enormous clinical potential. This article highlights significant advances made in biomaterial-based protein delivery strategies over the last 25 years and how they will influence research in the next 25 years. These advances will enable protein release rates to be tuned with increased flexibility to deliberately address the challenges of the dynamic injury environment and ultimately lead to better solutions for patients.

Targeted protein delivery: carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds.

Tissue engineering response may be tailored via controlled, sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional (3D) ice-templated collagen scaffolds. However, the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored. Here, we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide) microparticles. We probe the effects of subsequent N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride crosslinking on protein release, using microparticles with different internal protein distributions. Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug. The scaffolds display a homogeneous microparticle distribution, and a reduction in pore size and percolation diameter with increased microparticle addition, although these values did not fall below those reported as necessary for cell invasion. The protein distribution within the microparticles, near the surface or more deeply located within the microparticles, was important in determining the release profile and effect of crosslinking, as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold. Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release. Protein located within the bulk of the microparticles, was protected from the crosslinking reaction and no delay in the overall release profile was seen.

Conjugation of Anti-EpCAM Antibody on Alginate–RIP MJ-30 Nanoparticle through Carbodiimide Reaction as a Model of Targeted Protein Therapy

Ribosome inactivating proteins from Mirabilis jalapa L. (RIP MJ) has shown higher cytotoxic activity when being formulated as a nanoparticle. However, the selectivity of the delivery system is also an important aspect when it comes to cytotoxic cell therapy. Epithelial cell adhesion molecule (EpCAM) is a monomeric glycoprotein which is overexpressed in epithelial cancer cells. This study aim was to develop a model of targeted protein delivery system by formulating the base fraction of RIP MJ (RIP MJ-30) into alginate nanoparticles and conjugating it with anti-EpCAM antibody. RIP MJ-30 was formulated in to nanoparticle using alginate and CaCl2 as cross-linker. Optimization of conjugation reaction condition was done in the pH variation of 4.5, 5.5, and 6.5. The success of conjugation was analyzed qualitatively using native polyacrylamide gel electrophoresis (native-PAGE) method and BCA assay. The optimum formula of RIP MJ-30 nanoparticles was produced using 0.3% alginate and 0.2% CaCl2. Results indicated that optimum conjugation reaction was carried out at pH level of 5.5. The optimum native-PAGE condition was by using 8% polyacrylamide gel in duration of 6h. Characterization of nanoparticle resulted in particle size of 205.0nm, zeta potential of -6.9mV, entrapment efficiency of 71.11±4.84%, and conjugation efficiency of 89.55±6.18%. It was concluded that RIP MJ-30 was successfully formulated into alginate nanoparticle and conjugated to anti-EpCAM antibody through carbodiimide reaction using 1-ethyl-(dimethylprophilamine) carbodiimide (EDAC).

Controlled Epidermal Growth Factor Receptor Ligand Display on Cancer Suicide Enzymes via Unnatural Amino Acid Engineering for Enhanced Intracellular Delivery in Breast Cancer Cells.

Proteins are ideal candidates for disease treatment because of their high specificity and potency. Despite this potential, delivery of proteins remains a significant challenge due to the intrinsic size, charge, and stability of proteins. Attempts to overcome these challenges have most commonly relied on direct conjugation of polymers and peptides to proteins via reactive groups on naturally occurring residues. While such approaches have shown some success, they allow limited control of the spacing and number of moieties coupled to proteins, which can hinder bioactivity and delivery capabilities of the therapeutic. Here, we describe a strategy to site-specifically conjugate delivery moieties to therapeutic proteins through unnatural amino acid (UAA) incorporation, in order to explore the effect of epidermal growth factor receptor (EGFR)-targeted ligand valency and spacing on internalization of proteins in EGFR-overexpressing inflammatory breast cancer (IBC) cells. Our results demonstrate the ability to enhance targeted protein delivery by tuning a small number of EGFR ligands per protein and clustering these ligands to promote multivalent ligand-receptor interactions. Furthermore, the tailorability of this simple approach was demonstrated through IBC-targeted cell death via the delivery of yeast cytosine deaminase (yCD), a prodrug converting enzyme.

Cytosolic Protein Delivery: Programed Assembly of Nucleoprotein Nanoparticles Using DNA and Zinc Fingers for Targeted Protein Delivery (Small 52/2018)

Cytosolic Protein Delivery: Programed Assembly of Nucleoprotein Nanoparticles Using DNA and Zinc Fingers for Targeted Protein Delivery (Small 52/2018)

Programed Assembly of Nucleoprotein Nanoparticles Using DNA and Zinc Fingers for Targeted Protein Delivery.

With a growing number of intracellular drug targets and the high efficacy of protein therapeutics, the targeted delivery of active proteins with negligible toxicity is a challenging issue in the field of precision medicine. Herein, a programed assembly of nucleoprotein nanoparticles (NNPs) using DNA and zinc fingers (ZnFs) for targeted protein delivery is presented. Two types of ZnFs with different sequence specificities are genetically fused to a targeting moiety and a protein cargo, respectively. Double-stranded DNA with multiple ZnF-binding sequences is grafted onto inorganic nanoparticles, followed by conjugation with the ZnF-fused proteins, generating the assembly of NNPs with a uniform size distribution and high stability. The approach enables controlled loading of a protein cargo on the NNPs, offering a high cytosolic delivery efficiency and target specificity. The utility and potential of the assembly as a versatile protein delivery vehicle is demonstrated based on their remarkable antitumor activity and target specificity with negligible toxicity in a xenograft mice model.