Animal venoms constitute a vast source of bioactive molecules with pharmacological properties which have evolved for millions of years to efficiently interfere with the essential physiological processes of the prey ^[1][2][5,6]. Due to their toxicity and general biological effects, these natural resources have long attracted scientific interest ^[3][7]. Animal venoms are complex mixtures of peptides, proteins, salts, neurotransmitters, nucleosides, and other compounds ^[4][8]. Approximately 90% of the dry weight of some venoms (like that of snakes) consists of proteins and peptides. Among the protein classes, there are phospholipases A2, proteases (serine and metallo), l-amino acid oxidase, hyaluronidases, cysteine-rich secretory proteins (CRISPs), phosphodiesterases, nerve growth factors (NGFs), etc. Among the bioactive peptide classes, there are antimicrobial, analgesic, hypotensive, cytolytic/cytotoxic, cell-penetrating, anticancer (antitumoral) peptides, neuropeptides, and others.

The advances in the search for active biomolecules based on animal venoms indicates their effectiveness, which is mainly against cancer and diseases caused by microorganisms. ^[5][6][7][9,10,11]. However, it is well known that biomacromolecules from animal venoms, mainly proteinaceous ones, have low bioavailability or stability in biological media or in vivo treatments. In this context, few studies have reported the structure-function relationship of these biomacromolecules and drug delivery strategies ^[8][12].

Nanobiotechnology is a modern tool that helps to protect these compounds and even directs the target destination of the biomacromolecules, enhancing their activity and effectiveness ^[9][13].

2. Nanosystems and Nanocarriers

Nanobiotechnology combines two important branches of modern science, namely nanotechnology and biotechnology. On the one hand, nanotechnology satisfactorily meets the physicochemical characteristics of protection and drug delivery. Its particles are on a nanoscale (size around 1–1000) and have pharmacological properties of high interest worldwide ^[10][14]. This is because nanocarriers optimize bioavailability, reduce adverse effects, and increase drug activity ^[11][15]. On the other hand, biotechnology is a science that studies the application or management of a living organism to improve or obtain a beneficial product ^[12][16]. This technological combination helps to obtain new compounds with high prospects on the market and benefits for the population ^[13][17]. In recent years, the macromolecules of animal venoms have been reported to have outstanding effects, such as antivenom, anticancer, and anti-inflammatory properties, among others ^{[14][15][16][17]}[18,19,20,21]. However, this potential and activity are often limited by the different factors of the biological environment to which they are exposed, and many times these macromolecules lose activity or diminish their effect. For example, some proteolytic enzymes present in the blood may denature or break peptide and protein bonds. Snake venoms are a complex mixture of biomacromolecules, containing different components, with more than 90% of their dry weight corresponding to proteins. Among the protein content of these venoms, there is a wide range of enzymes and peptides ^[18][22]. These molecules are responsible, alone or in synergism, for the physiological effects observed after animal envenomation. Venoms have been applied as therapeutics since antiquity, and technological advances have allowed the isolation and determination of the structure and properties of their components. As a result, their potential as new medicines has been explored in many fields. Macromolecules from animal venoms are considered high-profile biotechnological products and, combined with the protection of their components conferred by nanotechnology, highly impressive results are expected in the coming years.

2.1. Nanoliposomes

Nanoliposomes (NLPs) are drug delivery and carrying systems made up of phospholipidic layers that cover an aqueous fraction ^[19][60]. NLPs have many advantages, but their main importance lies in their high biocompatibility, friendliness to the environment, and non-toxicity ^[20][61]. NLPs are vesicles that can be classified and distinguished depending on the method of preparation and size. They can be unilamellar (small unilamellar vesicles—SUV, 20–80 nm; large LUV, 80 nm–1 μm; and giant GUV, >1 μm), multilamellar (MLV, >400 nm), or multivesicular (MVV, >1 μm) ^[21][62]. Vesicle preparation requires careful formulation since the size of the particle differs when performed randomly. It was previously reported that the thin film hydration method would be an adequate formulation for antimicrobial peptides (AMPs) such as bacteriocins. This technique develops heterogeneous-MLV and, with assisted energy, it is possible to obtain LUVs or SUVs ^[22][63]. NLPs encapsulated with AMPs such as nisin, a cationic peptide, could confer, through its electrostatic properties, greater affinity for anionic lipids such as dicetylphosphate, or, through a hydrophobic affinity, zwitterionic neutral lipids such as phosphatidylcholine. Therefore, a better encapsulation efficiency is achieved in relation to anionic phospholipids such as phosphatidylglycerol ^{[23][24][25][26]}[64,65,66,67]. Depending on the lipid and the electrostatic effect produced by the AMP, the stability of the NLP could be altered, and consequently better stability and zeta potential higher than 30mV would be achieved. The antimicrobial activity could also be related to the net charge of the lipid system formed, since the charge of the bacterial membrane is negative, which could repel the accommodation of NLP in pathogenic bacteria; this detail must be taken into account when formulating new antimicrobial agents ^[27][68]. Another peptide, Vicrostatin (VCN), has been encapsulated with NLPs. VCN is a peptide originating from the fusion between echistatin (Echis carinatus) and contortrostatin (Agkistrodon contortrix contortrix venom) fractions ^[28][29][69,70]. Vicrostatin is described in the literature as a potential anti-angiogenic and pro-apoptotic tumor agent ^[30][71]. The trapping of animal venoms has become relevant due to the condition of transport to specific cancer cells, and it was shown that this type of venom-NLP nanocomposite achieves better anticancer effects in various cell lines ^[31][72]. Considerable application advances are being made in venom-NLPs in the biopharmaceutical industry since NLPs are already in the preclinical and clinical phases, mainly for the treatment of cancer ^[30][31][32][25,71,72]. Specifically, regarding the encapsulation of components from snake venoms with NMPs, some peptides can be cited. Melittin is the main component of the venom of the European bee Apis mellifera. It is a 26-mer peptide, originating from a two-step enzymatic cleavage of a preproprotein. The first cleavage removes an N-terminal signal peptide, while the second cleavage removes an anionic sequence responsible for inactivating melittin through interaction with C-terminal cationic residues ^[33][34][73,74]. Nanotechnology and intratumoral injection greatly decrease the required dose of melittin peptide, which generates cost reductions and significantly decreases its cytotoxicity, since melittin by itself is highly toxic and an uncontrolled dose could cause irreparable damage. A study revealed that α-melittin-NPs resulted in delayed tumor growth and even in the complete regression of the distant tumor. Furthermore, α-melittin-NPs have several strengths, including simple preparation, good stability, and a lack of side effects ^[35][27].

2.2. Silica Nanoparticles (SiNPs)

SiNPs are nanosystems with high drug delivery efficiency that vary in size from 10 to 500 nm. These nanosystems are the most widely studied and scaled for drug administration. Non-porous SiNPs are a type of SiNPs, characterized mainly by their large surface area, easy functionalization, and biocompatibility. One of the systems best adapted to peptides, proteins, and DNA is MSNs, since their ordered and porous design allows a greater load of the bioactive compound for therapeutic application ^[36][86]. MSNs are nanoparticles ranging in size from 2 to 50 nm, generated mainly by the Stober method or its modifications, and they are widely used on account of their simple manufacture, low cost, and availability on an industrial scale ^[36][37][86,87]. They can be classified according to their nano-application as MSNs sustained drug delivery systems (MSNs-SDDSs) which exhibit deliberate drug release, responding automatically to the conditions of the medium or a concentration gradient; and as MSNs stimuli-responsive controlled drug delivery systems (MSNs-CDDSs), which instead release the drug at a controlled level in response to a physical or chemical stimulus ^[38][88]. SBA-15 nanoparticles have a non-toxic and adjuvant effect which modulates the immune system in mice by inducing the production of IgG2a and IgG1 isotypes. This is beneficial when it comes to anticancer, anti-inflammatory, or antimicrobial treatments, especially in people with compromised immune systems ^[39][90]. This type of biotechnological formulation using SBA-15 is generally carried out under acidic conditions and using a source of silica, like tetraethoxysilane (TEOS), in combination with an amphiphilic triblock copolymer such as Pluronic 123 composed of poly (ethylene oxide) (PEO) and poly (propylene oxide) (PPO), i.e., PEO-PPO-PEO triblock ^[40][91]. There are several examples of encapsulation of molecules derived from animal sources with SiNPs. Exenatide (39-amino-acid peptide), a molecule present in the saliva of the lizard from the Gila monster (Heloderma suspectum), is very similar to the incretin hormone glucagon-like peptide-1 (GLP-1). Clinical studies and clinical experience with exenatide have shown a significant reduction in HbA1c, fasting and postprandial glucose and a marked reduction in body weight in patients with type 2 diabetes mellitus. Animal studies have shown an improvement in blood function, beta cells, and an increase in beta-cell mass after treatment with exenatide ^[41][93]. GLP-1R agonists are promising anti-obesogenic and anti-dyslipidemia drugs in the early stages of obesity, in which the integrity of the nervous system has not been affected ^[42][94]. Currently, exenatide is available commercially under the names Byetta™ and Bydureon™.

2.3. Metallic Nanoparticles

Metallic nanoparticles (MNPs) are nanosystems that incorporate the use of metals to enhance the antimicrobial activity of different bioactive compounds, the best known of which are gold (AuNPs) and silver (AgNPs). MNPs can currently be obtained by various methods; however, it is considered that these methods may cause environmentally toxic effects because the metals are difficult to degrade. For this reason, green synthesis processes are recommended ^[43][109]. Some venom components from vipers, such as crotamine, a peptide composed of 42 amino acids, with 4.8 kDa and isoelectric point around 10.8, were first isolated from the venom of the Argentinean rattlesnake C. durissus terrificus ^[44][110]. It is a small basic myotoxin containing three disulfide bonds (Cys4-Cys36, Cys11-Cys30, and Cys18-Cys37) ^{[45][46][47][48]}[111,112,113,114] which acts on sodium and potassium channels ^[49][50][115,116], exerting myotoxic effects. Crotamine induces microorganism death by peroxidation and lipid oxidation of target proteins, determined by substances reactive to thiobarbituric acid and sulfhydryl groups, respectively ^[51][117]. Antibacterial and antifungal activities were evaluated in ATCC strains and clinical isolates, showing promising results ^[6][10]. Likewise, antitumor treatment was confirmed after the successful treatment of a mouse model grafted with a subcutaneous melanoma tumor ^[52][118]. Other biological activities, such as anti-leishmanial, anti-helminthic, and antimalarial effects, were reported ^{[53][54][55][56]}[119,120,121,122]. Crotamine-AuNPs via a PEG linker were synthesized (NP size 14.6 nm); in vitro assays confirmed the internalization of these nanoparticles into living HeLa cells. AuNPs are playing a progressively more significant role in multimodal and multifunctional molecular imaging to detect and treat diseases, such as early-stage cancer ^[57][38].

2.4. Polymeric Nanoparticles

Polymers are linear or branched conformations of covalently linked monomers ^[58][123]. The nanoparticles obtained from this material are relevant because the loading content of the bioactive is efficiently high, and in most cases, the use of surfactants is necessary since many of the drugs are usually hydrophobic ^[59][124]. Chitosan nanoparticles (ChitNP) are nanosystems that have been used in formulations for treatments involving mucoadhesion. Chitosan is a biopolymer that has a positive charge, and an affinity is generated by an electrostatic bond, targeting the mucosa, which is negatively charged; in addition, it has biocompatible characteristics and the ability to permeate the intestinal barrier, which stimulates the passage of molecules through the paracellular and transcellular pathways ^[58][123]. Although this polymer has certain limitations due to its low solubility at pH > 7, it is considered a good releaser of active compounds in the duodenum for gastrointestinal formulations. In vitro and in vivo studies have shown a non-toxic response, but the results of clinical studies that fully corroborate its application are still lacking ^[60][126]. ChitNP showed promising antimicrobial as well as anti-biofilm properties and is used mainly for the development of new nanostructured products, as it can destabilize the bacterial membrane and inhibit protein synthesis and mRNA transcription ^[10][14]. Poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) are currently highly studied nanosystems. PLGA is an FDA-approved polymer due to compound protective potential, biocompatibility, and biodegradability ^[61][127]. However, PLGA-NPs have not shown good drug delivery properties in specific cells/proteins since the release of the compound occurs in an uncontrolled manner. Generally, this type of nanoformulation is accompanied by coatings or conjugates that can help this type of targeting, such as polyethylene glycol (PEG), polydopamine (PDA), hyaluronic acid, and folic acid, among others ^[62][63][128,129]. In addition, the cost-benefit ratio of using this polymer does not make it attractive since PLGA is relatively expensive and the particle size is around 500 nm. However, some non-toxic chemical agents can increase encapsulation rates and the release period can be carried out for as long as one month, as with bee venom (Park, Min-Ho 2016). Hamzaoui and Laraba-Djebar ^[64][43] reported obtaining and trapping the venom of Cerastes cerastes (horned viper) and Vipera lebetina in PLGA-NPs used as a vaccine; these reptiles are native to North Africa and the Middle East, respectively, and have a high bite incidence rate in the region. These PLGA-NPs (EE 84.1%) were administered intranasally, generating antibodies and high Th2 values, which favored humoral immunity; the NPs provided venom tolerance of 6LD50 and 5LD50, respectively ^[64][43]. Finally, another toxin isolated from the Leiurus quinquestriatus scorpion venom is chlorotoxin (CHTX), which is a neurotoxin composed of 36 aminoacids that was first isolated by DeBin et al. ^[65][133] and is described as a calcium channel blocker. Selective and specific binding of CHTX to glioma cells was demonstrated by immunochemical techniques, and radiolabeled CHTX was shown to bind only to cells in a glioma tumor of a mouse xenograft model ^[66][67][134,135]. CHTX-morusin-PLGA NPs exhibited excellent pharmacological properties against the treatment of glioblastoma or brain cancer with a mechanism directed towards tumor cells. Likewise, there is evidence that crossing the blood-brain barrier is a great challenge that NPs manage to meet ^[68][45].

3. Nanobioconjugations

Nanosystems loaded with animal venoms tend to increase the therapeutic effect, while bioconjugation can further increase this effect ^[69][70][40,150]. The bioconjugation of nanoparticles is generated by embedding a molecule in another nanomolecule. For this purpose, ligand techniques can be applied, the most promising ones being click chemistry application techniques such as Diels-Alder. The SBA-15 system loaded with melittin uses the effect known as trigger-responsive valves, and shows the opening of the silica-based porosity at pH 5.5, which is the pH of cancer cells. This system was conjugated with 3-mercaptopropyltrimethoxysilane (MPTS), then with 2,2-Bis (N-maleimidoethyloxy) propane (MK-Linker, acetal), and finally with melittin, when a Cys was added to the sequence (d-amino acids) of melittin at the N-terminus ^[71][92]. Melittin-large pore MSNs capped by electrostatic interaction with β-cyclodextrin (β-CD)-modified polyethyleneimine (PEICD) were synthesized. It was observed that the MSN/pore size ratio was 90–110/7–10 nm, offering this nanobioconjugate (with assembly and loaded nanosystem) efficacy in the controlled release of AMP and/or high and low molecular weight drugs. In addition, this product was able to reduce the formation of biofilm (between 20 and 30%) and eliminate 24 to 27% of the total bacteria. However, the scholarauthors also explained that when using melittin-MSN combined with NPs-Magnetics of MnFe₂O₄ loaded with ofloxacin, previously synthesized by thermal decomposition, they were able to reduce the biofilm formation of P. aeruginosa PAO1 cells by up to 97% and eliminate 100% of this bacterium ^[72][151]. Some toxins, such as NN-32 (from the Indian cobra venom, Naja naja), have remarkable anticancer activity, but its high cytotoxicity does not make it an attractive macromolecule. However, when it was conjugated with AuNPs, the cytotoxicity decreased considerably without affecting its antitumor activity; this property was evaluated by means of in vitro (lymphocytes and macrophages) and in vivo (mice) assays ^[70][150]. In breast cancer cells, NN-32 incorporated into AuNPs induced apoptosis and discontinued tumor proliferation ^[70][150]. In addition, Lycosin-I from Spider (Lycosa singorensis) functionalized in AuNPs showed specific antitumor activity as an excellent nanocarrier to obtain novel drugs using these toxins ^[73][58]. Maurocalcine from Scorpio maurus palmatus functionalized with AuNPs showed selectivity for three different cell lines such as human epithelial-like HeLa, MDA-MB-231 (human breast adenocarcinoma), and A431 (epidermoid carcinoma cell), which allows nanosystems to be adapted to antitumor activity ^[74][59].