Emerging Landscape of Supercharged Proteins and Peptides for Drug Delivery

Abstract

Although groundbreaking biotechnological techniques such as gene editing have significantly progressed, the effective and targeted transport of therapeutic agents into host cells remains a major obstacle to the development of biotherapeutics. Confronting the unique challenge posed by large macromolecules such as proteins, peptides, and nucleic acids adds complexity to this issue. Recent findings reveal that the supercharging of proteins and peptides not only enables control over critical properties, such as temperature resistance and catalytic activity, but also holds promise as a viable strategy for their use in drug delivery. This review provides a concise summary of the attributes of supercharged proteins and peptides, encompassing both their natural occurrence and engineered variants. Furthermore, it sheds light on the present status and future possibilities of supercharged proteins and peptides as carriers for significant biomolecules in the realms of medical research and therapeutic applications.

With the advancement of biotechnology, large biological molecules such as proteins and nucleic acids have gained increasing prominence in disease treatment.¹ A primary focus in contemporary pharmaceutical and biomedical research revolves around achieving intracellular delivery of these biomacromolecules, aiming to expand the applications and target sites for protein-based therapeutic, prophylactic, and diagnostic macromolecules. Several strategies have emerged to address this objective, including the utilization of positively supercharged (PSC) cell-penetrating proteins, various nanocarriers, and the genetic incorporation of positively charged peptide modules that facilitate protein internalization, known as Cell Penetrating Peptides (CPP) or Protein Transduction Domains (PTD).²⁻⁴ These biomacromolecules penetrate cells to execute their biological functions and are often linked covalently or noncovalently to carriers. This multifaceted approach enhances the potential for the precise and effective delivery of therapeutic agents within cells.

A family of naturally occurring proteins known as supercharged proteins have net charges that are abnormally high, either positive or negative (>1 net charge unit/kDa molecular weight). The number of cationic amino acids, such as lysine, arginine, and histidine (Lys/Arg/His), is correlated with the positive charge of these proteins. Proteins and peptides having a high net surface charge are known as supercharged proteins and peptides.⁵ Its excellent water solubility and antiaggregation capacity make it very applicable in the fields of material science, biology, and medicine. Biomolecules including DNA, siRNA, and proteins can be carried into cells by proteins and peptides with abnormally high net charges. These molecules can also be engaged in gene editing.^2,4 These proteins and peptides offer the following benefits over conventional transporters: excellent cell spectra, high transport efficiency, and minimal cytotoxicity.

Positively charged supercharged proteins and peptides form multivalent complexes with negatively charged biological macromolecules through electrostatic interactions, resembling the assembly of chromatin in organisms with histones and negatively charged DNA.⁵ Genetically engineered or electrostatically interacting proteins and peptides with exceptionally high net charges have the capacity to deliver plasmid DNA, siRNA, and proteins to mammalian cells, thereby exerting their biological functions (summarized in Figure 1).⁵ Research suggests that artificially modified supercharged proteins can exhibit an abnormally high net charge on the protein surface while retaining the basic functions of the original protein,^6,7 offering new characteristics such as antiaggregation properties,⁸ thermal stability,⁹ and strong membrane-penetrating abilities,¹⁰ opening new avenues for protein function modification. This study provides a comprehensive review of the distinctive characteristics of supercharged proteins and peptides with unusually high net charges and explores their applications as carriers for biomolecular penetration.

Figure 1.

Schematic diagram of supercharged protein and peptides’ application. Supercharged proteins and peptides, through deliberate engineering or modification, find diverse applications. In pharmaceuticals, they contribute to the development of enhanced therapeutic agents and drug design, offering improved delivery, stability, and efficacy. In materials science, these biomolecules play roles in biosensors, material synthesis, and the creation of advanced materials. Biomedical imaging benefits from their application as delivery agents, aiding in precise visualization in medical imaging. Protein and enzyme engineering utilizes modified proteins to boost catalytic activity for industrial processes. In vaccine development, they enhance antigen presentation for improved immune responses.

Supercharged Proteins

Supercharged Proteins in Nature

Charged Proteins in Nature

In nature, proteins are generally electrically neutral, but approximately 5% of proteins carry a higher positive charge, participating in the normal biological activities of organisms. As shown in Table 1, these proteins are involved in processes such as genome replication (Sperm proteamine P3, Sperm protamine P1), transcription (30S ribosomal protein Thx), and protein synthesis (60S ribosomal protein L41), maintaining the basic life activities of organisms.

Table 1. Summary of Positively Charged Proteins in the UniProt Database.

In 2011, Cronican et al. discovered a series of naturally occurring supercharged proteins that not only penetrate cell membranes but also carry biological macromolecules into cells to exert biological functions.¹¹ These supercharged proteins include HRX, the c-Jun bZIP domain, eotaxin3, HBEGX, N-DEK, and N-HGF (Summarized in Table 2). N-DEK is an autoantigen related to transcription, chromatin structure, and mRNA processing. Studies suggest that DEK, stimulated by IL-8 and secreted by macrophages, can act as a chemotactic factor, attracting neutrophils, CD8T target cells, and natural killer cells.¹² c-Jun protein participates in various cellular activities in the body, such as proliferation, apoptosis, and tumor development. c-Jun bZIP can bind DNA and regulate gene transcription. HBEGF and N-HGF are extracellular growth factors (summarized in Table 2). NEIL1 is a large molecular weight, highly charged protein. During enzyme-catalyzed biotinylation, this supercharged protein can deliver noncovalently bound chain-maleimide affinity tags to hamster kidney cells. Proteins with exceptionally high net charges are often found in the capsid proteins of viruses. The research team fused these supercharged proteins with red fluorescent proteins and Cre recombinase through genetic engineering, and the expressed fusion proteins could penetrate cell membranes and exert corresponding biological functions. Moreover, the internalization efficiency of supercharged proteins was 40 times higher than that of cell-penetrating peptides (TAT,¹³⁻¹⁵ Arg10, penetratin), and the recombination efficiency induced by Cre was significantly improved. The research team injected supercharged protein-Cre fusion proteins into the retinas, pancreas, and white adipose tissue of mice, discovering that functional proteins could be delivered to mammalian tissues to exert their effects.¹⁶

Table 2. List of Supercharged Proteins with Delivery Capacity.

Dengue virus C protein (DENV C) belongs to the family of flavivirus capsid protein family. It consists of 100 amino acid residues, and its three-dimensional structure is a dimer with 42 positive charges.¹⁷ Freire demonstrated that DENV C protein can not only penetrate cell membranes but also carry plasmids and siRNA into mammalian cells. However, the delivery mechanism of naturally occurring supercharged proteins is still unclear, and the modification of the protein supercharges may alter the properties and activities of proteins. This deserves further in-depth understanding and exploration to promote the application of supercharged proteins as carriers for biologically large molecules in medical research and disease treatment.¹⁸

Engineered Supercharged Proteins

In 2007, David et al. conducted a study on the surface structure of green fluorescent protein (GFP) molecules.¹⁹ They replaced solvent-exposed nonconserved amino acids with basic or acidic amino acids, resulting in proteins with abnormal charges, known as “supercharged proteins”. Without altering the fundamental functions of the original protein, increasing the charge on the protein surface changes its physicochemical properties (summarized in Figure 2).²⁰

Figure 2.

Summary of the 3D structures of engineered supercharged proteins as outlined in published references. Supercharged GFP (green fluorescent protein) refers to an engineered version of the naturally occurring GFP, often modified by introducing additional charged residues (positive: Lys, Arg, negative: Asp and Glu) to its amino acid sequence. Surface residue supercharging to the cellulase protein, Escherichia coli cytochrome b562, widely known proteins GST, Streptavidin, and SpyCatcher protein,^108,109 typically through genetic engineering, to enhance its performance or specific properties.

David et al. expressed a total of 28 supercharged green fluorescent proteins in E. coli, ranging from −30 to +48 in net charge.^21,22 They found that as the net charge increased, the membrane-penetrating ability of supercharged proteins exhibited an S-shaped growth pattern. The membrane penetration of supercharged proteins was found to depend on endocytosis and macropinocytosis. Compared to cationic peptides, supercharged proteins demonstrated a higher membrane-penetrating efficiency. Further research focused on 36+GFP due to its stable physicochemical properties and physiological functions. 36+GFP could adsorb to the cell membrane surface (which carries a negative charge) through charge interactions, efficiently passing through the cell membrane. Research also indicates that 36+GFP proteins can form stable complexes with siRNA, delivering the complexes into various mammalian cell lines, including HeLa (cervical cancer cells), IMCD (mouse inner medullary collecting duct epithelial cells), 3T3-L (mouse embryonic fibroblasts), PC12 (rat adrenal medullary chromaffin cell line), and Jurkat (acute T-cell leukemia line). Among these, IMCD, 3T3-L, PC12, and Jurkat cells showed resistance to the cationic lipid transfection reagents. SiRNA entered the cells and inhibited the expression of target genes.²³

Cronican et al. found that fusing 36+GFP with the target protein mCherry (a mutant variant of red fluorescent protein) using genetic engineering techniques resulted in proteins that could successfully penetrate cells, and red fluorescence could be observed inside the cells.²⁴ Additionally, fusing 36+GFP with Cre recombinase and using the expressed fusion protein to mediate the transfection of the pCALNL-DsRed2 plasmid into HeLa cells revealed that the plasmid could enter the cells and express itself. Subsequently, Zuris et al. found that cationic liposomes could deliver anionic supercharged proteins (−30GFP) and the Cre recombinase, TALEN (transcription activator-like effector nucleases), and Cas9:sgRNA complexes into HeLa cells, achieving efficient protein delivery and gene editing. Compared to traditional delivery methods, the delivery efficiency of the Cas9:sgRNA complex increased to 80%, with greater specificity.²⁵ However, a significant challenge faced by protein therapy is nuclear escape.²⁶ Wang et al. developed lipid nanoparticles capable of encapsulating anionic biologically supercharged GFP (−27) and Cre recombinase or Cas9:sgRNA electrostatic complexes for delivery into mammalian cells and the mouse brain.²⁷ After lipid degradation, improvements were observed in nuclear escape and cargo release, effectively providing gene recombination or gene editing.

Furthermore, it has been discovered that supercharged green fluorescent proteins also exhibit antiviral and antitumor effects (summarized in Table 3). In 2018, Motevalli et al.²⁸ and Shabazi et al.²⁹ found that +36GFP could deliver HPV16E7 DNA and proteins to HEK-293T cells. In in vivo experiments, complexes formed by +36GFP-E7 DNA and +36GFP-E7 protein protected mice from TC-1 tumor attacks. In 2019, Vahabpour et al. observed a significant reduction in HCV and HSV replication in HEK-293T cells infected with HCV and Vero cells infected with HSV when +36GFP was present.³⁰ The transfection rate of the HPV16E7 antigen increased, and HIV replication decreased by 75%. In in vivo experiments, +36GFP-E7 protein inhibited the growth of HPV-related tumors in mice.

Table 3. List of Supercharged Protein Applications in Biotherapeutics Delivery.

Name	Cargo	Model	Application	Ref
–30GFP	Cre recombinase	HeLa	protein delivery	(25)
	transcriptional-activator-like effector (TALE) proteins	HEK293T	transfection of Cas9 and sgRNA expression plasmids	(25)
	CRISPR-Cas9: sgRNA	U2OS EGFP reporter cell and mouse inner ear	gene editing	(25)
+36GFP	Cy3-GAPDHsiRNA	HeLa, PC12, IMCD, 3T3-L, and Jurkat T	gene silencing	(23)
	plasmid of β-galactosidase	HeLa and PC12	plasmid transfection	(23)
	ubiquitin	HeLa, 3T3, and baby hamster kidney cells	protein transduction	(24)
	pCALNL-DsRed2 plasmid	HeLa and NIH-3T3	plasmid transfection	(24)
	Cre recombinase	mouse retina		(24)
	mCherry	HeLa, baby hamster kidney cells, NIH 3T3, IMCD, and PC12	protein transduction	(24)
	HPV16 E7 DNA and HPV16 E7 protein	HEK-293T, A549, TC-1, and C57BL/6 tumor mice model	plasmid transfection and protein transduction	(28,29)
	pDsRed plasmid	MCF7 and HSC-T6; CD1 mouse	plasmid transfection	(110)
+36GFP-Dot1l	pDsRed plasmid	MCF7 and HSC-T6; CD1 mouse	plasmid transfection	(110)
aurein1,2-+36GFP	Cre recombinase	inner ear of live mice	protein transduction	(111,112)
B1 protein	nef-vpu-gp160-p24 and nef-vif-gp160-p24	HEK-293T; BALB/c mice	plasmid transfection	(113)
supercharged coiled-coil protein	β-galactosidase, siRNA, and doxorubicin	MC3T3-E1 and MCF7	transfection	(114,115)
+scGFP	CRE-encoding plasmid and biovesicles-packaged floxed reporter pDNA-scBVs	Ai9 mice	transfection	(116)
+9GFP	IGF1	bovine cartilage explant and chondrocyte, and human cartilage explant	protein transduction	(117,118)

In the same year, Chen et al. first applied +36GFP to photothermal cancer therapy. They combined it with sulfonated polyphenylalanine (SPNPG) to enhance its water solubility and induce a negative surface charge, forming nanocomplexes through electrostatic interactions with supercharged proteins (SPNPG/ScGFP).³¹ Experiments showed that the introduction of ScGFP greatly improved the absorption efficiency of photothermal therapy. Both in vitro and in vivo results indicated effective inhibition of melanoma growth. Additionally, ScGFP served as a fluorescent imaging agent, allowing for visualized treatment, highlighting its significant potential in the biomedical field. In 2020, Cao et al. used CRISPR/Cas technology to integrate the recombinant eGFP-TRAIL gene into the 19th chromosome of neutrophils.³² During the process of trapping cancer cells, TRAIL selectively induced apoptosis in tumor cells and the positively charged surface modification of eGFP increased DNA binding. Among them, + 36GFP showed the best results, representing a novel approach to cancer treatment.

In addition to GFP protein, other proteins can also have their properties altered through supercharging.^33,34 Liu et al. applied the same method as GFP to supercharge glutathione transferase (GST) and found that its catalytic activity remained unchanged.¹⁹ However, when heated to 100 °C and then cooled, it retained 40% of its catalytic activity. A different human enteropeptidase variant, when subjected to supercharging, demonstrated heightened solubility and stability. This suggests the potential application of this method for enhancing enzyme characteristics.^35,36 The Whitehead team observed that introducing a negative charge to cellulase resulted in reduced lignin adsorption, consequently lowering the cellulase production costs. While pressurized cellulase exhibits diminished catalytic efficiency compared to the wild-type counterpart, adjusting salt concentration has been identified as a means to restore its activity, presenting novel avenues for exploration.³⁷ In a noteworthy development in 2023, DeChellis et al. achieved a pressurized cellulase with a net charge range of −52 to +37. Surprisingly, they discovered that mutations in the carbohydrate binding module (CBM) domain were pivotal in enhancing cellulase activity on cellulose, underscoring the role of proteins in modulating enzyme activity through charge regulation via pressurization.³⁸ Subsequently, Simeon et al. discovered a B1 protein composed of 244 amino acids with a net charge of 43.³⁹ This protein not only inhibited the replication of hepatitis C virus (HCV), a process related to its high charge, but also facilitated the delivery of proteins or nucleic acids into mammalian cells. Crooks et al. found that supercharging the surface glycoproteins of HIV increased the neutralization rate of neutralizing antibodies, providing a new method for vaccine design.⁴⁰ In 2021, Eagen et al. found that supercharging the fusion protein BRD4-NUT could recruit and activate a large amount of histone acetyltransferase (p300), forming acetylation foci. These foci enriched numerous proteins, referred to as mega-domain proteins (MDs).⁴¹ As superenhancers, MDs activated transcription in a BET-dependent manner, making the functional fusion protein BRD4-NUT useful in developing top-notch BET inhibitors. In 2022, the Cavazzini team designed a supercharged thermophilic thioredoxin (+21PfTrx),⁴² which, although enhanced the membrane-penetrating ability of fusion proteins, tended to internalize into the nucleus. Nevertheless, derivatives of +21PfTrx were discovered (containing three tandem repeats of the HPV16-L2 B cell epitope), which induced immune responses more quickly and exhibited superior immunogenicity, making it a promising vaccine carrier. In 2023, the Chrysanthou research group found that supercharged albumin not only retained the surface tension of microdroplets but also could be coupled with surfactants to achieve the physical cross-linking necessary for strong interfacial elasticity.⁴³ Thus, supercharged albumin can serve as a scaffold to promote the adsorption of extracellular matrix (ECM) proteins, holding significant importance in the culture of adherent cells, especially stem cells. Simultaneously, Pistono et al. found that supercharging virus-like particles (VLPs) increased their uptake by mammalian cells.⁴⁴ The common VLP, MS2 bacteriophage, showed a significant increase in internalization efficiency after supercharging, reaching 6–7 times that of wtMS2. Moreover, these variants were found to deliver monomethyl auristatin (MMAE) to neuroglial tumors, exhibiting therapeutic efficacy comparable to antibody–drug conjugates, providing a new approach for cancer treatment.

Supercharged Peptides

Inspired by natural supercharged proteins, supercharged peptides (SCP) are recombinantly expressed in E. coli. The high positive net charge of SCP is associated with a repetitive unit composed of five peptides (VPGKG). By varying the monomer length and repeating multiround connections, a series of SCPs with different numbers of repetitive units and chain lengths are generated.⁴⁵ In 2018, Yin and others identified a series of supercharged peptides (K1, K2, K3, and K4) through genetic engineering.⁴⁶ These SCPs can be fused with GFP using gene engineering methods, expressed, and purified into the corresponding fusion proteins. These fusion proteins can penetrate various cell types including HeLa cells, BEL-7402 liver cancer cells, A549 human lung cancer cells, PC12 rat chromaffin cells, HSC-T6 cells, and Jurkat T cells. Among them, K4-GFP has the highest transmembrane efficiency. Research has shown that K4 can penetrate the cell membrane, effectively escape from the endosome, and deliver proteins nontoxically to the cell nucleus. K4 remains stable in serum, exhibits low toxicity, and has low immunogenicity in vivo. However, K4 is applicable only to nuclear-targeted proteins and requires the construction of plasmids and protein purification, making the process complex. Subsequently, the research group introduced a non-natural amino acid at the N-terminus of SCP, containing a phenylboronic acid (PBA) side chain, named PBA-SCP. PBA-SCP can form complexes with cargo proteins through ionic interactions, nitrogen–boron salt coordination, and electrostatic interactions, simplifying the protein binding process. PBA-K20 can deliver GFP proteins to the cytoplasm and antibodies to the cytoplasm without affecting antibody functionality, targeting and labeling intracellular antigens.⁴⁷ Additionally, SCP is involved in gene editing, delivering Cas9:sgRNA complexes functionally to mammalian cells. Although pure SCP can enhance delivery efficiency, Cas9 cutting is suboptimal, possibly due to spatial hindrance of SCP. The group subsequently introduced a connectable disulfide cyclic peptide linker between SCP and Cas9, effectively improving gene editing in tumor cells.⁴⁸ SCP has also been found to be applied as a lubricant and adhesive in clinical settings. In 2020, Wan and colleagues synthesized supercharged peptides K72, K108, and K144 using genetic engineering.⁴⁹ They introduced two cysteines at both ends of SCP to generate K108cys and K144cys. Experimental results showed that K108cys effectively enhanced saliva biolubrication strength and prolonged the saliva lubrication time for dry mouth patients, increasing from 3.8 to 21 min. In 2021, Sun and colleagues synthesized a series of SCPs with different chain lengths and charges.⁵⁰ SCPs were found to strongly adhere to wounds when combined with synthetic surfactants through electrostatic interactions, providing a new approach for reshaping wound microenvironments and promoting rapid wound repair. Based on SCP, Pesce and others reported another type of elastin-like polypeptide (ELP).⁵¹ By introducing lysine or glutamic acid residues at the second position of the repetitive pentapeptide sequence (GVGVP) and positively or negatively supercharging these amino acids, they adjusted the overall charge of the protein. Experimental results showed that ELP could deliver GFP proteins into A549 cells, and delivery efficiency gradually increased with an increase of positive charge. The protein with the highest positive charge, GFP-K72, showed a 6-fold increase in uptake compared to unmodified GFP.⁵² Therefore, supercharged peptides are powerful carriers for delivering large molecules into cells, warranting further exploration and research. Additionally, Ma et al. used anionic supercharged peptides containing glutamic acid (GVGXG)n to predict and design a new class of protein biomaterials obtained through genetic engineering.⁵³ These biomaterials have promising prospects as H⁺ transport membranes in biosensors and microimplantable biofuel cells.

Cell-Penetrating Peptides (CPPs)

Cell-penetrating peptides (CPPs) are short peptides consisting of 5–40 amino acids that can penetrate the cell membrane and enter the cell’s interior. Research indicates that CPPs can carry siRNA, DNA, short peptides, and proteins into cells, exerting specific biological functions.²⁻⁴ They are characterized by a high content of basic amino acids, resulting in an overall positive charge. We define a supercharged cell-penetrating peptide (summarized in Table 4) when more than 50% of the amino acids in the CPP are cationic.

Table 4. List of Functional Delivery by Supercharged CPPs.

Name	Sequence	Net charge	Proportion of net charge	Cargo	Testing model	Ref
PDX-1	RHIKIWFQNRRMKWKK	+8	0.5	rPdx1Δ protein and green fluorescent protein	Hyperglycemia mice	(61)
CyLoP-1	CRWRWKCCKK	+5	0.5	pEGFP-nef-vpr-gp160-p24 and rNef-Vpr-Gp160-P24	HEK-293T	(62,119)
Scp01-b	VSRRRRRRGGRRRRGGGSYARVRRRGPRGYARVRRRGPRR	+21	0.52	GFP expression plasmid	Caski and HSC-T6	(120)
P1	KKKKKRFSFKKSFKLSGFSFKKNKK	+13	0.52	GFP expression plasmid	MCF7 and HSC-T6	(121)
P10	KKKKKRFSFKKSKLSGFSFKKNKK	+13	0.54	oleanolic acid	HSC-T6	(122)
hPP10	KIPLPRFKLKCIFCKKRRKR	+10	0.5	GFP, apoptin, GCLC, plasmid, siRNA, and PROTAC degrader	Caski, human peripheral blood lymphocytes, and mouse model	(123−125)
A2–17	LRKLRKRLLRLWKLRKR	+10	0.58	5(6)-carboxyfluorescein	CHO-K1 cells	(126)
MT23	LPKQKRRQRRRM	+7	0.58	apoptin	B16	(127)
DPV6	GRPRESGKKRKRKRLKP	+10	0.58	Cre recombinase	HEK-293 cells carrying the integrated Lox-TK-Lox-GFP reporter construct	(128)
AIP6	RLRWR	+3	0.6	fluorescein isothiocyanate	RAW 264.7, LoVo colon cancer cells, and zymosan-induced inflammation mouse model	(129)
DPV3	KKRRRESRKKRRRES	10	0.625	Cre recombinase	HEK-293 cells carrying the integrated Lox-TK-Lox-GFP reporter construct	(128)
Dot1l	KARKKKLNKKGRKMAGRKRGRPKK	+15	0.68	GFP expression plasmid and recombinant GFP protein	MCF7 and HCC-T6	(130,131)
hPP3	KPKRKRRKKKGHGWSR	+11	0.68	GFP, KLA, and NBD	Caski and HSC-T6	(132)
P2	RKRRQTSMTDFYHSKRRLIFSKRK	+10	0.41	HaloTag	HepG2 and HeLa	(131)
LDP-NLS	KWRRKLKKLRPKKKRKV	+12	0.7	β-galactosidase	HeLa	(133)
CVP1-N2	LKRLRRRYKFRHRRRQRYRRR	+15	0.71	red fluorescent protein (RFP) and apoptin	HCT116	(60)
R9F2C	RRRRRRRRRFFC	+9	0.75	PMO	HeLa and NIH3T3	(134)
Pep4 (CHAT)	CHHHRRRWRRRHHC	+11	0.78	pEGFP-N1 and pLuc-Cy5	MCF-7, MDA-MB-231, DU145, and PC-3	(135)
(KH)9-BP100	KHKHKHKHKHKHKHKHKHKHKKLFKKILKYL-NH2	+26	0.83	psfGN155-MxMT and psfGC155-MxMT	tobacco leaf cells	(136)
Tat	RKKRRQRRR	+8	0.88	siRNA	primary chondrocytes	(137)
LDP-NLS	CHHHHHRRRRRRRRRHHHHHC	+19	0.9	pEGFP-nef-vpr-gp160-p24 and rNef-Vpr-Gp160-P24	HEK-293T	(62,119)
polyarginines R8, R9	RRRRRRRR; RRRRRRRRR	+8; +9	1	siRNA	primary cultured neurons	(59)

CPPs, as carriers for delivering large molecules, typically exist in two forms: covalent and noncovalent binding. In 1988, Frankel and colleagues discovered the TAT peptide encoded by human immunodeficiency virus type 1 (HIV-1).⁵⁴ Subsequently, research groups reported a novel fusion protein linked to the TAT peptide of the p19 protein,⁵⁵ which could deliver siRNA into Huh7 cells, inducing gene silencing without cellular toxicity. Furthermore, research found that covalently formed fusion proteins with TAT could cross the blood–brain barrier for treating brain ischemic injuries, such as Bcl-xL,⁵⁶ GDNF,⁵⁷ and SOD,⁵⁸ showing excellent efficacy in a brain ischemia model.

Kumar and colleagues found that a chimeric peptide composed of polyarginine (Arg9) and rabies virus glycoprotein (RVG) could deliver siRNA into neuronal cells,⁵⁹ inducing gene silencing. When intravenously injected into mice, RVG-9R successfully delivered siRNA to neurons, causing specific gene silencing in the brain. This suggests that CPPs provide a new pathway for delivering biomacromolecules through the blood–brain barrier.

In 2020, Hu and colleagues identified a novel CPP-CVP1-N2 within chicken anemia virus. They discovered its ability to deliver red fluorescent protein (RFP) and apoptosis genes into cells, inducing cell apoptosis.⁶⁰ PDX-1, a critical transcription factor controlling pancreatic β-cell development, possesses a protein transduction domain for cellular entry.⁶¹ Recombinant PDX-1 was found to enter cells, improving the blood glucose conditions in diabetic mice.

Due to CPPs carrying cargo into cells through endocytic pathways, cargo is often trapped inside the endosomes, a major factor limiting CPP delivery efficiency. However, CPP-CyLoP-1 can enter cells at low micromolar concentrations and deliver EGFP-nef-vpr-gp160-p24 and rNef-Vpr-Gp160-P24 into HEK-293T cells, increasing cargo delivery efficiency.⁶²

While CPPs often exhibit poor pharmacokinetics, with most cargo being sequestered in endosomes, limiting their ability to deliver target molecules and exert biological functions, efforts have been made to enhance CPP escape from endosomes.^2,4,63 In 2017, Kristina and others designed dfTAT (TAT dimer).⁶⁴ When dfTAT and the target molecule reached late endosomes through coincubation, dfTAT promoted late endosomal membrane leakage, delivering the target molecule into cells. Additionally, the team found that increasing the number of arginines enhanced the peptide’s penetration ability, but lysines did not achieve the same effect.

In 2018, Brock et al. discovered that trimeric TAT (3TAT) had a stronger effect in promoting endosomal escape.⁶⁵ Compared with monomeric and dimeric TAT, 3TAT accumulated in the nucleus at a higher concentration, making it easier to reach the concentration threshold for membrane disruption.

Cationic Antimicrobial Peptides

Antimicrobial Peptides in the Human Body

Due to the serious threat to public health posed by antibiotic resistance resulting from antibiotic misuse, antimicrobial peptides are increasingly considered as alternatives to conventional antibiotics. Antimicrobial peptides, isolated from prokaryotes (bacteria) and eukaryotes (protists, fungi, plants, and animals), are essential components of the natural defense mechanisms of most organisms. These peptides, consisting of 8–50 amino acids, exhibit a positive net charge ranging from +2 to +9 due to the high content of lysine and arginine residues. They have a molecular weight of 2–10 kDa and contain 40% hydrophobic amino acids, rendering them amphipathic.⁶⁶

These peptides demonstrate broad-spectrum activity against both Gram-positive and Gram-negative bacteria, fungi, and parasites (summarized in Table 5). They play a crucial role not only in innate immunity but also in promoting inflammation, proliferation, wound healing, cytokine release, homeostasis, chemotaxis, and maintaining the balance between proteases and protease inhibitors.⁶⁷ Their ability to selectively recognize potential pathogens prevents or limits infections, making them an indispensable part of the body’s defense.

Table 5. List of Antimicrobial Peptides from Humans.

Name	Source	Residues	Net charge	Function	UniProt No.	Ref
hBD-1	kidney, skin, and salivary gland	36	+4	F, G+, G–, C	P60022	(138,139)
hBD-2	skin, lungs, epithelial cells, uterus, and salivary gland	41	+7	F, G+, G–	O15263	(140)
hBD-3	skin and salivary gland	45	+11	F, G+, G–	P81534	(141)
hBD-4	testicles, lungs, kidneys, and neutrophils	48	+7	G+, G–	Q8WTQ1	(142)
LL-37	skin and neutrophils	37	+6	F, G+, G–, P, C	P49913	(143−145)
HIS1	saliva from the submandibular gland and parotid gland	57	+8	F	P15515	(146)
HIS3	saliva	51	+12	F, G+, G–	P15516	(147)
HIS5	saliva	24	+5	anti-Candida albicans, anti-Cryptococcus, and anti-Aspergillus fumigatus		(148)
dermcidin	perspiration	47	–2	F, G+, G–	P81605	(149,150)
RNase 5	liver, skin, and gut	125	+11	F, G+	P03950	(151)
RNase7	urinary and respiratory tract, skin	127	+16	F, G+, G–	Q9H1E1	(152,153)
chemokine CCL20	skin	69	+8	F, G+, G–, P	P78556	(154)
chemokine CXCL9	blood	125	+22	G+, G–, P	Q07325	(155)

Delivery Functions of Antimicrobial Peptides

Scientists have discovered that some peptides exhibit both cell-penetrating peptide (CPP) characteristics, allowing them to penetrate mammalian cell membranes, and antimicrobial peptide (AMP) functions, enabling them to kill pathogenic bacteria. Moreover, literature suggests an overlap in the structure and mode of action between CPPs and AMPs.⁶⁸

For instance, the antimicrobial peptides Magainin2 and Buforin2 can enter HeLa and TM12 cells, and Buforin antimicrobial peptide can covalently attach to green fluorescent protein for delivery into cells.⁶⁹ Some CPPs can also enter bacteria to exert antimicrobial effects. Researchers found that CPP-Pep-1 can mediate elastin protein delivery into fibroblasts, treating skin aging.⁷⁰ Additionally, Pep-1, prone to substituting glutamic acid for lysine, yielded a new type of antimicrobial peptide (Pep-1-K) capable of resisting various pathogens, making it a promising candidate for local antimicrobial therapy.⁷¹

In 2002, scientists designed the amphipathic peptide ppTG20, demonstrating its ability to transfect plasmid DNA into various human and murine cell lines.⁷² Later, based on the structure–activity relationship of CPPs and AMPs, scientists replaced Phe and Trp with Arg in ppTG20, resulting in a ppTG20 analogue (P7) proven to have a bactericidal effect on Salmonella typhimurium,⁷³Escherichia coli ATCC25922,⁷⁴ and Candida albicans.⁷⁵ CPP-TP10, a transport protein analogue lacking 21 amino acids, inhibits the growth of Candida albicans and Staphylococcus aureus. pVEC, another cationic CPP, inhibits the growth of Mycobacterium avium at low micromolar concentrations without toxic effects on HeLa cells.⁷⁶ Therefore, antimicrobial peptides hold great potential as novel carriers for delivering large molecules into cells.

Biotechnological and Biomedical Applications of Supercharged Proteins

A multitude of applications in biotechnology and biomedical engineering have benefited from the creation of novel protein complexes. This includes the development of scaffold enzyme cascade reactions, drug delivery,⁷⁷ charge transport,²⁰ and the production of biosensors.⁷⁸⁻⁸⁰ Notably, research groups have explored the potential of supercharged proteins in various innovative applications.

One research team utilized +36GFP as a signal reporting factor, assembling it into complexes with negatively charged DNA through electrostatic interactions. The results indicated high specificity in DNA methylation detection.⁸¹ Moreover, +36GFP demonstrated a remarkable ability to penetrate cells and carry DNA, providing a new approach for targeted intracellular detection with broad prospects.

Wang et al. discovered that supercharged protein (ScGFP) can interact with graphene oxide (GO).⁸² The efficient quenching of ScGFP fluorescence by GO was exploited to develop a novel fluorescence biosensing system. James et al. devised a set of VX binding proteins incorporated into a pressurized scaffold DRNN (net charge 27). The scaffold’s capability to encapsulate all binding sites within the protein contributes to its high affinity and specificity, facilitating swift and precise detection of VX neurotoxins. This presents a novel approach for creating small molecule sensors.⁸³ Tang and colleagues devised a detection method based on ScGFP and cationic copolymers (CCP), mediating fluorescence resonance energy transfer between ScGFP and CCP in a complex.⁸⁴ This approach opens new directions for screening PARP-1 inhibitors and developing PARP-1-related anticancer drugs.

Furthermore, researchers employed ScGFP/GO as probes for detecting glycosaminoglycans (GAGs), achieving visualized detection at both the cellular and animal levels.⁸⁵ Supercharged proteins also serve as potential dual-reporter genes.⁸⁶ Wu utilized the cell-penetrating capability of +36GFP to design a short peptide binding to lanthanide elements for magnetic resonance imaging, serving as a cell imaging probe for disease detection.⁸⁷

Later, Ghaheh and colleagues discovered that supercharging enhances the fibrinolytic activity of plasma prothrombin while maintaining the stability of Reteplase,^88,89 a thrombolytic drug. This discovery opens new possibilities for the clinical treatment of myocardial infarction. Additionally, scientists found that supercharged green fluorescent protein (GFP) can alter enzyme catalytic activity. They covalently connected three ScGFP mutants (−30, 0, +36) with AdhD (thermostable ethanol dehydrogenase), revealing that the −30GFP complex exhibited the highest catalytic efficiency, followed by 0GFP, while +36GFP was unaffected.⁹⁰ This suggests a significant correlation between enzyme catalytic efficiency and the charge carried by supercharged proteins, highlighting supercharged protein fusion as a convenient method to modify enzyme microenvironments.

Moreover, supercharged proteins, acting as protein cages, play a role in the transport of biological macromolecules such as siRNA and proteins, yielding mature results.^91,92 Azuma et al. demonstrated the control of enzyme catalytic activity by packaging enzymes in protein cages.⁹³ They expressed a recombinant protein, the +36GFP-enzyme, through genetic engineering. This protein self-assembled with negatively charged protein cages, forming stable complexes. Katherine proposed a protein encapsulation system by incorporating +36GFP into a mutant AfFtn (E65R) (thermostable ferritin), providing high stability and potential protection against protein degradation, folding, and immune damage during transport of therapeutic cargoes.⁹⁴ Korpi and colleagues combined two natural protein cages, Pyrococcus furiosus (aFT) ferritin and cowpea chlorotic mottle virus (CCMV), with supercharged peptide K72 and recombinant protein GFP-K72.⁹⁵ They discovered the reversible self-assembly of these complexes, signifying their importance in maintaining the stability of large molecules.

Furthermore, Zhang et al. found a peptide soft material formed under external force by the complexation of negatively charged supercharged peptides and cationic surfactants containing azobenzene.⁹⁶ This discovery indicates that peptide soft materials, through their self-organization behavior under external forces, can record fingerprint information and individual recognition, offering an alternative to complex optoelectronic assistance. To freely transport small protein molecules into cells, Wang and others designed a peptide sequence (VPGXG)n, where X represents a variable charged amino acid.⁹⁷ By fusion of amino acids with the target protein, the high charge of SCP enhanced the driving force of the target protein through nanochannels, significantly improving the resolution of the measurement system. This SCP, by precisely controlling protein transport through nanochannels, holds tremendous potential for protein fingerprinting and proteomics applications.

In addition to enhancing protein stability, supercharging can also regulate the overall function of proteins. Teams have successfully applied supercharging to polymerases and other enzymes to enhance their functionality.^98,99 For instance, adding a fusion domain to the thermostable Bacillus subtilis DNA polymerase improved its performance in isothermal amplification detection. Further supercharging of this domain could increase the protein stability and diagnostic performance. Simon and colleagues discovered that proteins with extremely high net positive and negative charges could self-assemble into symmetric protein oligomers through their electrostatic interactions (summarized in Figure 3).¹⁰⁰ For instance, a 16-mer composed of two stacked octameric rings consisting of Cru+32 and GFP-17 provided new insights into the structures and materials in biological systems. Cummings and others found that positively charged supercharged proteins, when mixed with polyanions (DNA/RNA/PAA/PSS), mostly precipitated in a solid phase at the reaction midpoint.¹⁰¹ This transition between liquid–liquid or liquid–solid phases at different concentration ratios provides clues to abnormal coagulation processes observed in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).

Figure 3.

Supercharged proteins engineered for self-assembly enable the spontaneous generation of macromolecular structures. Supercharged proteins, meticulously engineered for self-assembly, exhibit the remarkable ability to autonomously generate intricate macromolecular structures. This innovative approach to protein design not only harnesses the intrinsic properties of these biomolecules but also opens new avenues in nanotechnology and materials science, offering the prospect of creating customized and functional materials with diverse applications. The tailored self-assembly of supercharged proteins represents a cutting-edge frontier in biotechnology, presenting opportunities for advancements in drug delivery, biosensors, and other fields where precisely structured macromolecules are pivotal.

Perspective and Conclusion

Supercharged proteins and peptides, characterized by remarkably high positive or negative charges, can engage in electrostatic interactions to form complexes with biopolymers carrying opposite charge. This holds tremendous potential for transporting DNA, siRNA, and proteins into mammalian cells through noncovalent or covalent conjugation (as illustrated in Figure 4). Such interactions facilitate essential functions like protein expression, gene silencing, and gene editing.¹⁰² However, challenges often arise when large molecules are delivered into the body or its cells. This is due to the obstacles that most proteins and biological macromolecules, such as DNA, must overcome to enter cells and carry out their biological function. These obstacles include serum interference, cellular internalization, and endosomal escape. Numerous scientists are attempting to overcome these obstacles.

Figure 4.

Supercharged proteins engineered as a versatile drug delivery vector through both covalent and noncovalent conjugation strategies. Supercharged proteins, strategically engineered for drug delivery applications, serve as versatile vectors employing both covalent and noncovalent conjugation approaches. Through covalent binding, these proteins establish stable linkages with therapeutic agents, ensuring controlled release and targeted delivery. Simultaneously, noncovalent conjugation mechanisms, such as electrostatic interactions or hydrophobic associations, enable dynamic and reversible connections, allowing for adaptable drug loading and release. This dual conjugation strategy enhances the versatility of supercharged proteins as drug delivery vectors, offering a flexible platform to address diverse therapeutic needs with precision and efficiency. The tailored engineering of these proteins presents a promising avenue for advancing the field of drug delivery, optimizing treatment outcomes through their unique and tunable conjugation properties.

Strategic mutations in proteins and peptides typically represent an effective approach to improving their properties, including bioactivity and stability. Some research groups have mutated the arginine and lysine residues in cationic cell-penetrating peptides (CPPs) to histidine residues, resulting in peptides with pH responsiveness. These peptides are electrically neutral under physiological conditions but become positively charged upon entering acidic endosomes. This enables them to interact with the endosomal membrane, facilitating the entry of cargo proteins into cells.¹⁰³ Another approach involves the fluorine modification of cationic polymers to enhance the stability of complexes. Experimental results show that fluorinated high polymers can transfect cells under very low nitrogen-to-phosphorus ratios, reducing cell toxicity caused by increased positive charges.¹⁰⁴

The application of supercharged proteins or peptides in the treatment of diseases involving large biomolecules brings about a renewed sense of hope. These agents show significant potential for intracellular medication delivery and gene therapy. Nevertheless, several challenges must be overcome before their clinical implementation. The lack of tissue and cell specificity in charged proteins or peptides results in nontargeted entry into cells, leading to unnecessary loss of biological function. A pivotal objective for future development is to improve targeting. Some research teams have identified short peptides, ranging from 3 to 14 amino acids, known as cell-targeting peptides (CTPs).^105,106 CTPs can facilitate targeted medication delivery by binding to receptors overexpressed on the surface of cancer cells. Despite their high specificity, CTPs face challenges, such as low oral bioavailability, poor stability, and a tendency to be cleared by the kidneys. Furthermore, there are few reported experimental results on the full therapeutic effects of supercharged proteins transporting drugs in vivo. While the probable uptake mechanism for these positively charged proteins involves an electrostatic interaction with surface glycosaminoglycans, followed by endocytosis, as demonstrated with a supercharged +36 GFP variant,¹⁰⁷ it is important to note that the cellular uptake pathways can vary due to the different shapes and sizes of particles. This diversity in uptake mechanisms may necessitate a more comprehensive evaluation before clinical applications, requiring additional resources for a thorough assessment. Questions remain about the potential toxicity of supercharged proteins as transport tools as well as the toxicity and immunogenicity of degradation products after cellular internalization. Further optimization is required to improve their applicability, especially considering the potential long-term effects in vivo, and we believe that with the continued development of biotechnologies there will be breakthroughs, gradually resolving the issues related to the targeted delivery of biomacromolecules mediated by supercharged proteins and peptides. This, in turn, will provide further support for the use of supercharged proteins as drug carriers in disease treatment.

Data Availability Statement

Data sharing is not applicable to this article as no data sets were generated or analyzed during the current study.

No sources of funding were received for the preparation of this article.

The authors declare no competing financial interest.