Precision Targeting of Toxins for Enhanced Therapeutic Efficacy and Safety

1.1. Venom peptides in biomedical research

Natural products have been used in medicine since ancient times and they are still holding relevance today, as many pharmaceutical compounds originate from plants ^[3]. Some examples include: digoxin, isolated from foxgloves (Digitalis sp.), morphine and its analogues derived from poppies (Papaver somniferum), vincristine and vinblastine from Madagascar periwinkle (Catharanthus roseus) and paclitaxel from yewspecies (Taxus sp). In addition, many of the well-known antimicrobials, as well as immuno-modulators and other drugs have been developed from microorganisms and fungi ^{[1, 2, 3]} . Moreover, some products obtained from animals have also found their use in the medical field, even though many of them may be outdated in the present times. Even though the beginnings of studies into venoms from animals can be dated back to 17th-18th centuries, it is only from the 20th century onward that these studies gained any significance owing to technological developments ^[4]. The venom produced by an animal is a complicated blend of Peptides and proteins are significantly larger molecules than small organic compounds and require delivery directly to the victim via a sting or bite. In most cases, they can only be used for their effect if given parenterally, but there are exceptions. Considering that peptide toxins are quite fragile, chemical and biological approaches were not applicable to the investigation of their properties as they were for plant-made stable molecules ^{[5, 6, ]}. As science moved forward, it was possible to develop highly accurate methods for analysis such as liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy, which enabled a much deeper examination of proteins and peptides. This included the isolation, purification, identification of structure and bioactivity of those. Nowadays, the fields of proteomics, genomics, and transcriptomics are instrumental in the research of toxins made in venom. Venomous toxins are normally very effective biologically and have a great affinity toward particular protein molecules (receptors, ion channels, enzymes) in other organisms, being quite specific down to particular receptor subtypes. The composition of venoms is sometimes extremely complicated and incredibly diverse ^{[8, 9]}.  There are some kinds of organisms that produce an enormous variety of peptide toxins. In many cases, toxins of venom itself appear to be mutant forms of native proteins carrying out biological activity. Because of the decline in the discovery rate of small molecule drugs over recent years, the attention paid to peptides and biological drugs has significantly increased. Due to great structural diversity and bioactivity of animal venoms, one may consider this type of natural substances rather unexploited pharmaceutical "mine," full of drug candidates for further investigation ^[10]. To date, five FDA-approved drugs have already been developed on the basis of animal venom. Another promising agent based on animal venom components is Cenderitide, a recombinant naturopathic peptide produced from human Carburettor peptide and Dendroaspis natriuretic peptide (DNP), the latter isolated from green mamba. It has already been tested in clinical trials as a prospective heart failure treatment agent. Moreover, due to great selectivity of certain toxins, such as connotations, they have been extensively studied to understand the mechanisms underlying action of certain receptors and ion channels. Consequently, apart from being directly applied for drug discovery and development, certain venom toxins or their derivatives due to extremely high affinity to certain biomolecules can be used as target agents in novel drug. ^[11]

1.2. Targeted drug delivery

Non-specific adverse effects/toxicity as well as non-desirable therapeutic efficiency because of poor pharmacokinetics are some of the major drawbacks that pharmaceuticals have in their current form. Many of these concerns can be addressed using approaches to achieve targeted drug delivery. In general, targeted drug delivery is categorized as either passive or active targeting. Passive targeting involves the selectivity of drug accumulation being dependent on Properties related to physiology or anatomy of the target tissue are some examples include utilization of the compromised nature of blood vessels in tumors, such as use of cyclophosphamide based on the metabolic features of cancer.  The compound is active under the condition of low aldehyde dehydrogenase concentration, which is typical for cancer cells.Active targeting, on the other hand, involves the functionalization ofdelivery systems using ligands that bind specifically to cell receptors,i.e., those with a high affinity towards ion channels or enzymes. Thus, targeted drug delivery technologies are still only achieving limited success, with passive targeting resulting in more achievements: this encompasses many nanomaterial drug products that exploit physiological tropism (such as liposomes, albumin particles, lipid nanoparticles/LNP vaccines), reviewed in total with more than 60 FDA-approved nanoparticles and 15 FDA-approved liposomal drug products. ^[12,13].

1.3. Peptides as targeting ligands

The comparison between different types of active targeting ligands is shown in Fig.1. Among those molecules which were studied in the course of searching for the targeting ligands, all of them possess some pros and cons.Small molecules, for instance, are considered to be very stable and membrane permeable. Nevertheless, the selectivity for target tissues remains low for them. As far as antibodies are concerned, which seem to be the most effective at present, they demonstrate very specific binding to the target molecule. In contrast to small molecules, antibodies show high affinity to the target molecule, yet a lot of problems can still be defined concerning the antibodies. Peptides have the same structure as proteins; yet, they are smaller molecules with molecular weights from 500 to 5000 Da, placing them between large proteins and small molecules. In this sense, peptides represent an intermediate level combining the best features of both types of molecules. For example, peptides have high specificity and high binding affinity toward the targets ^{[14, 15]}.

Table 1: FDA approved drugs that originate from animal venom toxins

ACE – angiotensin-converting enzyme, GLP-1R – glucagon-like peptide-1 receptor, gp IIb/IIIa – platelet glycoprotein IIb/IIIa receptor.

Fig. 1: summary of advantages and disadvantages of commonly used active-targeting ligands. Scheme based on the information in review articles Produced in BioRender

.1.3.1. Venom Peptides vs. Antibodies as Targeting Ligands

Unlike the large mAbs (~150 kDa), the size of venom peptides is usually < 5 kDa. In general, the smaller size makes penetrated into interstitial spaces more likely in comparison to mAbs that tend to remain perivascular. Numerous venom peptides have been shown to be highly specific and selective modulators of subtypes of various receptors/ ion channels that can serve as a means for tissue or disease specific homing where the target receptors/channels are overexpressed (i.e., integrins or ion channels). On the other hand, mAbs have the capacity to target a wide variety of molecules located in the extracellular matrix and on the surface of cells. However, they are unable to penetrate into channel pores and serve as modulators ^[15]. Thus, in this case, both types of targeting molecules represent complementary approaches rather than competitors. They do not compete in terms of binding similar targets since there is no duplication of efforts and both expand the pool of potential targets within the druggable space. With respect to stability, sulfiderich venoms (knottins, cyclized peptides) are highly stable against heat and enzymatic degradation, thus enabling them to withstand the conditions within biological fluids even when they are large molecules. On the other hand, unmodified venom peptides could be susceptible to proteases unless they are designed for resistance (cyclization, PEGylation) ^{[15, 16]}.

1.4. Venom peptide sources and diversity

Peptides that can be used as targeting agents in drug delivery systems may be obtained in many different ways. Some methods include isolating appropriate peptides through high-throughput screening of large peptide libraries, which can be done using phage display screening technique or chemical libraries such as the OBOC library. On the other hand, peptides that exhibit biological activity in nature, where they have been designed to perform certain functions in organisms for reasons such as food gathering, protection, competition, or physiological functions, can also be identified. ^[17] Evolutionarily, venom is a convergent phenomenon found in multiple groups of animals that are not closely related to each other, and many different toxins have been discovered in the animal kingdom. Typically, specific toxins, which are structurally and functionally alike, can be found among a group of organisms that are closely related to each other. Venom peptides' evolutionary adaptations are primarily determined based on their functionality and purpose. For instance, venom used for defensive purposes has been modified to be the most painful to ward off potential attackers. In contrast, venom used for predatory purposes tends to be lethal to the prey animals. Therefore, their actions and physiological functions are significantly different between the two classes of animals. Fig 2 shows some of the molecular targets of venom peptides for

TD ^[21]

Table 2: Comparison of venom peptides vs. antibodies as targeting ligands.

ADCs – antibody-drug conjugates, mAbs – monoclonal antibodies, NPs – nanoparticles, PEG – polyethylene glycol.

It was previously thought that only the lizard species belonging to genusHeloderma are venomous; however, it is now known that other Anguimorphyllizards such as the varanids have venom glands, are capable of producingvenom and belong to the recently introduced group of reptiles called Toxicofera. Though less studied than their snake counterparts, lizard toxins have a broad spectrum of biologic activities, among which are antihypertensive, neurotoxic (helofensins), as well asplatelet aggregation inhibitory activities, and many more ^{[21, 22]}. Also, the venom of wasps, ants and bees includes several types of toxic peptides (for instance aculeotoxins, myrimecins and others) that have allergenic and cell-disrupting properties as well as disulfde-rich neurotoxins that affect voltage-gated ion channels and have, for the reason that numerous hymenopterans use their encountering venom as a defensive mechanism, been modified to prepare pain and inflammation. In addition to those examples ofinsect venoms, there are caterpillars of the lepidopteran species Lononia obliqua whose venom includes serine proteases,factor X and prothrombin activators. Contact with these caterpillars’ bristles will result in coagulopathy, which is often fatal. Venoms of centipedes have not yet been studied well, but they contain many unique peptides that do not resemble any other peptides, for example, helical arthropod-neuropeptide-derived (HAND) toxins. ^{[22, 25]}

Fig. 2: A schematic representation of the main molecular targets for venom peptides in various conditions.

 MMP-2 – matrix metalloprotease-2, GLP-1R – glucagonlike peptide-1 receptor, NMDA – N-methyl-D-aspartate, NAcHR – nicotinic acetylcholine receptor, CTX – chlorotoxin, TFTs – three-finger toxins. Image created in BioRender.

2. OVERVIEW OF THE CURRENT RESEARCH ON VENOM PEPTIDE-BASED TARGETED DELIVERY SYSTEMS

2.1. Cancer targeting

2.1.1. Chlorotoxin

Chlorotoxin (CTX) is a 36-amino acid peptide extracted from the venom of the deathstalker scorpion (Leiurus quinquestriatus) (Fig. 3). The name of the toxin came from its initial observation, in which it was found to inhibit small-conductance chloride channels in colonic epithelial cells. However, the most intriguing feature of this toxin has been revealed by its capacity for selective binding to glioma cells with extraordinary affinity while sparing normal brain tissue. In addition to gliomas, CTX also binds preferentially to other malignant tumours, expanding the range of possible applications for this toxin. A variety of molecules can be used as targets for CTX, such as matrix metalloprotease-2 (MMP-2), annexin A2, neuropilin-1 (NRP1) and CLC chloride channels. The existence of the "true" receptor for CTX remains a matter of debate, but there is sufficient evidence for all of the above-mentioned candidates. MMP-2 appears to be the most probable choice.It is known that glioma is a particularly lethal brain tumor, where all the existing treatment strategies are rather inefficient ^[29,33].

Fig. 3: Sequence and disulfide bond arrangement of chlorotoxin, redrawn from Sharma et al. Image created in BioRender.

The variety of delivery systems examined for modification by CTX is indeed quite remarkable in many studies. Table 3, 4, and 5 show some of the examined delivery systems. Without question, it seems like CTX is the most investigated peptide in the realm of venom peptides. Nonetheless, it does seem that even though there are many successful studies on CTX so far, success rate has not yet reached perfection. So far, only evidence from preclinical stages on active targeting through CTX has been gathered ^{[1, 20, 30]}.

Table 3: Examples of CTX-functionalized nanoparticle systems for targeted therapy and imaging of cancer

CTX – chlorotoxin, DODAP – 1,2-dioleoyl-3-dimethylammonium-propane, DSPC – 1,2-distearoyl-sn-glycero-3-phosphocholine, DSPE – 1,2-Distearoyl-sn-glycero3-phosphorylethanolamine, DTPA –diethylenetriaminepentaacetic acid, GFP – green fluorescent protein, HPAO – 3-(4 hydroxyphenyl)propionic acid-Nhydroxysuccinimide ester, HSPC – hydrogenated soybean phosphatidylcholine,mal – maleimide, mPEG – methoxypolyethylene glycol, MR – magnetic resonance, NIR – near infra-red, NP – nanoparticles, PEG – polyethylene glycol, PEI – polyethylene imine.

Nanoscopic imaging systems that use other types of inorganic components have also been developed. Research has also been conducted on polymeric NPs with imaging agents attached to them. In one instance, Huang et al. synthesized an MRI contrast agent composed of dendrigraft poly-L-lysine labeled with CTX and containing a DTPA-Gd complex. The CTX-labeled nanoscopic agent demonstrated signal intensity for imaging of liver cancers and gliomas.

Table 4: Other ctx-functionalized delivery systems for cancer therapy and diagnostics.

CTX – chlorotoxin, IgG – immunoglobulin, MR – magnetic resonance, NIR – near infra-red, SPECT – single-photon emission computed tomography. Various types of polymeric and metal-oxide (mostly iron oxide) nanoparticles, alone and in combination with each other, have been utilized for the preparation of drug delivery systems targeting delivery of CTX. For example, the authors Mu et al. have developed iron oxide nanoparticles carrying CTX and gemcitabine via hyaluronic acid, as well as nanoparticles attached with CTX and a cyclodextrin molecule loaded with paclitaxel and 1 adamantenemethylamide-fluorescein. On the other hand, Stephen et al. used PEGylated, CTX and O6-benzylguanine-conjugated iron oxide nanoparticles for sensitizing glioma cells toward temozolomide, thereby overcoming their resistance toward the latter. ^[33]

This particular delivery system indeed showed improved effectiveness of temozolomide, 3 times enhanced median survival rate of mice and reduced toxicity of benzylguanine within the system. Yoo et al. also developed such a delivery system but with O6-methylguanine methyltransferase-silencing siRNA. There has also been research conducted on development of polymers for glioma gene therapy. Thus, for instance, Huang et al. synthesized nanoparticles of poly(amidoamine) (PAMAM) carrying genes on their surface and CTX via PEG. The DNA plasmid integrated into the nanoparticle carried the gene of tumor necrosis factor-related in any case, while the prospects might seem promising indeed, up until now there was only preclinical data demonstrating the effectiveness of such systems. Many times, the research was limited to only in vitro testing. It has been going on for more than two decades already since the beginning of the development of CTX. Still, at this time in 2025 none of the CTX-modified nanoparticle systems has even gotten to the clinical trials stage. Nanoparticles could provide for such property, but it still needs to be investigated thoroughly. Most of the tests done in vivo were conducted in rodents with a tumor xenograft model created. That does not really prove anything concerning their capability of crossing the BBB. ^{[34, 35]}.

2.1.2. Radiolabeled CTX analogues

The most interesting compound used in this approach is 131ITM-601, i.e. an analog of CTX radiolabeled with iodine-131 radioisotope. 131ITM-601 has been proposed as a substance for intracavitary application post glioma resection, i.e. as radiotherapeutic or radiographic agent. An in vivo experiment using glioma-bearing mouse model revealed pronounced selectivity in accumulating the agent in tumor cells. Specifically, radiation exposure measured in the tumors was 18-45-fold higher compared to most of the other body tissues (except brain, kidneys, and stomach), while still much lower compared to the latter three. 131ITM-601 has moved to the clinical trials stage. In Phase I trial the safety, pharmacokinetics and dosimetry of a single injection of 131ITM-601 intracavitation 2 weeks after cytoreductive craniotomy were evaluated in 18 adult glioma patients. The results showed no toxicity up to dose-limiting concentration and accumulation at target site and quick elimination of unbound peptide from circulation. ^[35]

Table 5: Delivery systems using ctx derivatives or structural analogues for cancer therapy and diagnostics.

BBB – the blood–brain barrier, BmK CT – Buthus martensii Karsch. chlorotoxinlike toxin, CTX – chlorotoxin, NIR – near infra-red, NPs – nanoparticles, PAMAM – poly-amidoamine, PEI – polyethylene imine, SPECT – single-photon emission computed tomography Moreover, some of the patients experienced the amelioration of the disease, and so, it might prove the possible therapeutic effect of the drug and the need for further phase II trials. A phase I/II trial was designed to evaluate the appropriateness of using this drug for the determination of glioma extent using MR/SPECT imaging in cases of high-grade recurrent glioma. According to the findings, the drug was not considered optimal for imaging because of its poor resolution. Additionally, the necessity of the intracranial administration of the drug made the researchers believe that the use of 131I-TM-601 was inappropriate since it could not get through the blood-brain barrier. Several more phase I and phase I/II studies seem to have been performed; however, their status was listed as “unknown” while one study was terminated without the publication of any results. There were no publications regarding 131I-TM-601 within the last 15 years. So, there were no definite reasons for failure but it might be doubted that this drug would be used again in the future. ^[36,44]

3. SNAKE VENOM TOXINS

3.1. Snake venom disintegrins. Snake venom disintegrins are peptides that interfere with integrins – cell membrane receptors involved in numerous physiological processes such as cell adhesion, haemostasis and immune responses, among others. The majority of snake disintegrins contain a common element in their structure – the Arg-Gly-Asp or RGD motif, which is considered to be the binding site for integrins. This motif also occurs in naturally-occurring integrin binding proteins. The contribution of other peptide residues located around the RGD motif, as well as the importance of peptide structure, is demonstrated by various potencies and integrin-binding properties of the peptides, and by the fact that some of the disintegrins function only when dimerized, whereas others can act as monomers. With regards to medical research, the most important target for drug development based on snake venom disintegrins is integrin αIIbβ3, or glycoprotein IIb/IIIa receptor (gp IIb/IIIa). This protein takes part in platelet aggregation, and therefore drugs blocking this receptor would be able to prevent blood clotting. Two FDA approved antiplatelet drugs have been obtained through disintegrins – tirofiban and eptifibatide ^[37].  Because many integrins play key roles in metastatic spread of cancer, tumor invasion and angiogenesis, the use of disintegrins as anti-cancer drugs has also received attention. It is indeed true that integrin receptors are often cited as one of the major targets for cancer-specific delivery, with the RGD tripeptide or its analogs having already been utilized for such purposes. However, in this article, we shall confine ourselves only to those disintegrins found in snake venom. Cancer-specific delivery systems using snake venom disintegrins are tabulated in Table 6 ^{[37, 38]}.

Table 6: Other venom peptide-functionalized delivery systems for cancer therapy and imaging

DMPC – 1,2-dimyristoyl-sn-glycero-3-phosphocholine, DOTA – 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid, DSPE – 1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine, DTX-1 – dendrotoxin-1, NAcHR – nicotinic acetylcholine receptor, NIR – near infra-red, PEG – polyethylene glycol, PET – positron emission tomography.

3.2. Neurotoxins: Several nicotinic acetylcholine receptors blocking neurotoxins from snake venom have been considered for use against the brain, nervous system or muscles, and also cancer cells overexpressing the receptors. They belong to 3 finger toxins (3FT) because of the specific loops observed in the molecule structure. Being involved in nerve impulse transduction and contraction of skeletal muscles, these molecules generally cause neuromuscular paralysis. Moreover, they are very potent neurotoxins. Liu et al. constructed an imaging reagent consisting of carboxylated nanodiamonds (cND) and α-bungarotoxin (α-BTX) – an α7-NAcHR blocking three-finger neurotoxin from venom of the multi-banded krait Surprisingly, no covalent linkages were used. The binding was realized through electrostatic interaction between negatively charged cND and positively charged BTX. The complexes were capable of binding α7-NAcHRs present in Xenopus laevis oocytes and human lung cancer cells and allowed visualizing them using confocal fluorescence microscopy. Nevertheless, the ability of the construct to be used in vivo seems doubtful considering the extremely high toxicity of α-BTX.

Fig. 5: Sequences and disulfide bond arrangement of contortrostatin (a), echistatin (b) and their recombinant fusion product vicrostatin (c)

4.  OTHER TOXINS

4.1. Conotoxins

 Conotoxins are highly diverse small and disulfide-rich pharmacologically active peptides isolated from the venom of cone snails (Conus sp.). At the current stage, only a tiny portion of these peptides and their pharmacological activities has been fully identified. Their high potency and highly selective affinity to biological targets make conotoxins interesting as potential candidates for targeted drug delivery systems development. So far it appears that α−conotoxins, known to target nicotinic acetylcholine receptors (NAcHRs), have been researched in this context. On the basis of the knowledge of α7 NAcHR overexpression in certain tumors, Mei et al. developed polymeric PEG-DSPE (1,2-distearoyl-sn-glycero3-phosphorylethanolamine) micelles conjugated with α-conotoxin ImI, isolated from the venom of Conus imperialis, as targeted carriers for cancer therapy.  In vitro, those nanoparticles were able to selectively recognize A549 lung cancer and MCF-7 breast cancer cells through interaction with α7-NAcHRs.

4.2. Bee venom peptides: Venom derived from honeybees (Apis mellifera) has been widely employed in various types of medicine, both alternative and conventional. As regards modern medicine, compounds isolated from bee venom continue to remain relevant in this respect, due to the amount of research on their possible therapeutic application. For instance, melittin – a cytotoxic peptide – might have some limited application as a targeting agent: Yu et al. suggested application of melittin in immunomodulatory nanoparticles, aimed at targeting and activating liver sinusoidal endothelial cells (LSECs) in order to achieve suppression of tumour metastasis formation in liver.  ^{[38, 45]}.

5. INSULINOMA TARGETING

5.1. Exendins

Exendins are peptide toxins, discovered in the venom of the North American genus of lizards, Heloderma. The best examples of such compounds are exendin-3 from the venom of Mexican beaded lizard (H. horridum), and exendin-4 from the venom of Gila monster (H. suspectum). Exendins are unique compounds, because of the high degree of structural homology with the human glucagon-like peptide-1 (GLP-1) – incretin hormone known to participate in digestion and insulin secretion (see Fig. 6). Initial in vivo experiments showed significant and long-lasting hypoglycemic effect of exendins. Moreover, exendin-4, especially, is significantly more resistant to cleavage by dipeptidyl peptidase-4, compared to natural GLP-1, as well as exhibiting a longer half-life time.

Fig. 6: Amino acid sequence comparison of exendins and related physiological peptides: a) exendin-4,  b) exendin-3, c) glucagon-like peptide-1, d) glucagon. Sequence of exendin-4 and homologous parts in other peptides are highlighted in magenta.

It should come as no surprise that interest was piqued in the possibility of developing exendin-4 as a pharmaceutical drug. Indeed, in 2005, exenatide (exendin-4) received FDA approval for use as an insulin-releasing agent to help control type II diabetes in individuals who do not respond to metformin and sulfonylurea treatment options. The mode of action of exenatide can be described as potent stimulation of GLP-1 receptors (GLP-1R), located primarily in the pancreas, among other effects, which leads to insulin secretion. In spite of the approval of exenatide, studies continued to be conducted on the exendins, revealing that exendins could serve as therapeutic agents in some other conditions.

5.2. Radiolabeled exendin conjugates for PET/SPECT imaging. Many pre-clinical in vivo studies were done using various types of exendin conjugates for imaging purposes, mainly based on exendin-4, occasionally on exendin-3, and even exendin(9-39), the latter one beingm an analogue with antagonistic activity to GLP-1R. The list of these compounds is shown in Table 7. Chemically, these molecules are formed by chelator molecule holding radioisotope of metal, coupled to the peptide through linker. Often extended termini (additional amino acid residues at the end of the peptide) serve as the binding sites. One of the first studies in this field and concerned the design, synthesis, and testing of in vivo 111In-[DTPA]labeled exendin-4 conjugate as an insulinoma detector ^[45].

Table 7 Summary of radiolabeled exendin conjugates for GLP-1R-targeted SPECT and PET imaging

CT – computed tomography, MR – magnetic resonance, PET – positron-emission tomography, SPECT – single-positron computed tomography

6. TARGETING THE BRAIN AND THE NERVOUS SYSTEM

6.1. CTX

In addition to targeting cancer cells, chlorotoxin (CTX) was also investigated for other purposes - including targeting the brain. The systems utilizing CTX for that purpose are presented in Table 10. described use of CTX modified liposomes for therapy of Parkinson's disease. This system was designed based on the reported capability of CTX to bind to microvascular endothelial cells which could potentially result in increased permeability of BBB and b metter delivery of drugs. In a mouse Parkinson's disease model, it was shown that the CTX modified and levodopa carrying liposomes resulted in an increase in Substantia nigra dopamine concentrations and amelioration of some neurological symptoms compared to the control group. Wang et al. investigated the autocatalytic polymeric nanoparticles carrying various substances aimed at increasing the permeability of BBB, such as lexiscan ^{[1, 25, 32]}. They discovered that attachment of CTX to the nanoparticle increased its delivery by a factor of 1.9. MiniCTX3 is the shortened and modified version of CTX designed by DiazPerlas et al. which proved to be stable in human serum, retained the ability to target certain sites and showed higher efficacy in crossing BBB in vitro as well as in transporting cargo through it. In another research, Han et al. used CTX and lexiscan in combination to design nanoparticles transporting Nogo-66 receptor antagonist NEP1-40 for the treatment of brain ischemic stroke. This design was used since it was discovered that in ischemic environment in brain there is upregulation of MMP-2.

6.3. Three-Finger Toxins

Another approach for enhancing the ability of BBB to permit passage is the targeting of particular receptors that are abundantly expressed in the brain, thus enabling receptor-mediated transport. Some neurotoxins that target NAcHRs have been studied for optimizing the BBB-permeation of CDX, a peptide that consists of 16 amino acid residues derived from the second loop of candoxin, which is a 3FT in the venom of the Malayan krait (refer to Figure 7). In this study, the researchers conjugated CDX to paclitaxel-loaded polymer micelles (polyethylene glycol-polylactic acid PEGPLA). It was shown that CDX micelles enabled drug delivery to the brain and suppressed the proliferation of glioblastoma cells in mice, thereby significantly increasing their lifespan compared to controls. conjugated CDX to red blood cell membrane-coated, doxorubicin-loaded poly-lactic co-glycolic acid (PLGA) nanoparticles. Such an approach showed an increased tumor accumulation of this system in the glioma mouse model, along with an increased lifespan of mice bearing glioma tumors, compared to the control group. ^{[ 41, 45]}

Fig. 7: Sequence and disulfide bond arrangement of a) candoxin, b) CDX

6.3.1. Bee venom peptides

Bee venom peptide apamin and its derivations are substantially used for CNS targeting due to their capability to increase blood – brain hedge (BBB) permeability. Apamin- grounded nanoparticles have shown picky accumulation in the spinal cord and bettered recovery in injury models. A crucial outgrowth, MiniAp- 4, is a more stable, less poisonous interpretation that retains apamin’s parcels while enhancing BBB penetration. MiniAp- 4 effectively delivers loadings (e.g., fluorescent labels) across the BBB and shows strong brain and excrescence targeting, especially in glioblastoma models. Structural variations, like replacing proline with  5,5- dimethylproline, may further ameliorate BBB crossing effectiveness. ^[46].

6.3.2. Peptides from Tarantula Venom

Spiders' venom peptides are known to act as potent agonists against diverse types of ion channels. They might therefore be considered promising agents for targeted drug delivery. reported the case of μ-TRTX-Pn3a, which is a tarantula (Pamphobeteus nigricolor) Nav1.7 blocker. (Sharma et al., 2020) Modification (addition of one glycine residue) at the N-terminal part of the peptide allowed for simple chemical conjugation with another molecule with the help of sortase A. Such modifications might find applications in targeted therapy or development of imaging agents. As an example, they have developed a conjugate with FLAG peptide, which might serve as a tool to visualize tissues with peptide accumulation via immunofluorescence. Another example is a Nav1.7-blocking toxin Hs1a, isolated from the Chinese bird spider Haplopelma schmidti ^[46].

Fig. 8: Sequences and disulfide bond arrangement of a) apamin, b) MiniAp-4