Lipid-Based Nanovesicular Drug Delivery Systems

In designing a new drug, considering the preferred route of administration, various requirements must be fulfilled. Active molecules pharmacokinetics should be reliable with a valuable drug profile as well as well-tolerated. Over the past 20 years, nanotechnologies have provided alternative and complementary solutions to those of an exclusively pharmaceutical chemical nature since scientists and clinicians invested in the optimization of materials and methods capable of regulating effective drug delivery at the nanometer scale. Among the many drug delivery carriers, lipid nano vesicular ones successfully support clinical candidates approaching such problems as insolubility, biodegradation, and difficulty in overcoming the skin and biological barriers such as the blood–brain one. In this review, the authors discussed the structure, the biochemical composition, and the drug delivery applications of lipid nanovesicular carriers, namely, niosomes, proniosomes, ethosomes, transferosomes, pharmacosomes, ufasomes, phytosomes, catanionic vesicles, and extracellular vesicles.


Introduction
Despite relevant technological improvements, developing an effective and safe drug can be a complex, low success rate, time-consuming, and costly practice. As reported on the official webpage of the US Food and Drug Administration (FDA), only a small number of treatment tools (active molecules, nanoparticles, and so on) proposed as skilled medical products, after early testing, result as eligible for further study. In 2020, the FDA's Center for Drug Evaluation and Research (CDER) authorized 53 novel therapeutics, more than double what happened from 2006-2010. More in details considering the three major therapeutic areas, the new approved drugs are 18 (34%) cancer products, 8 (15%) Neurology products, and 6 (11%) infectious diseases treatments. The average projected peak sales of a just approved drug in 2020 was about USD 700 million, and this is below a long-term average of USD 1.3 billion and a median of USD 500 million [1].
The constant development of technologies and materials resulting from the collaboration between sectors such as bioengineering, physics, chemistry, materials science, pharmacology, and not least medicine, has allowed the advancement of increasingly efficient drug delivery tools. Researchers and clinicians from all over the world daily pursue the design and implementation of increasingly personalized, safe, and cheap care solutions as new pharmacologically active molecules and nanoparticles. Recently, the application of nanoparticles (NPs) has been established to develop drug delivery efficiency. Nanomaterials generally refer to a material characterized by having at least one dimension in the nanometer scale (1-100 nm) [2], include nano-drug delivery systems that thanks to their morphological, optical, mechanical, and electrical characteristics can improve

Proniosomes and Niosomes
Niosomes and proniosomes are LNV systems characterized by distinctive amphiphilic structures able to improve poorly soluble drugs bioavailability. Their uniqueness is in having a nonionic surfactant backbone while their multilamellar and unilamellar vesicles structures appear similar to that of liposomes [6] (Figures 1 and 2). thanks to their morphological, optical, mechanical, and electrical characteristics can im prove drugs' stability and solubility by extending their blood circulation time and enhanc ing their delivery efficiency.
Among the already numerous nanoscale DDCs, nanovesicles represent highly-prom ising effective approaches to setting up therapies against cancer, inflammation infection and degenerative disorders.
In this review, we described the most modern lipid-based nanovesicular systems whether they are of biological or synthetic origin, used for the most distinct biomedica and clinical applications. We left liposomes, already the subject of numerous and recen scientific publications, out of the topics covered in this review, to make room for othe lipidic nanovesicles, perhaps less known, but increasingly the target of studies for drug delivery applications such as niosomes, proniosomes, ethosomes, transferosomes, phar macosomes, ufasomes, phytosomes, and catanionic vesicles. Last, but certainly not least the type of Lipid NanoVesicles (LNV) discussed in this review are the extracellular vesi cles (EVs) and their increasingly wide application as DDC of inorganic NPs, drugs, and nucleic acids. For each type of LNV category covered by the discussion, we provided an updated table listing in a very detailed way, the biochemical composition of each vesicle its cargo, and the application for which it has been designed and studied referring to the in vitro and in vivo drug delivery applications of the last 10 years.

Proniosomes and Niosomes
Niosomes and proniosomes are LNV systems characterized by distinctive am phiphilic structures able to improve poorly soluble drugs bioavailability. Their unique ness is in having a nonionic surfactant backbone while their multilamellar and unilamella vesicles structures appear similar to that of liposomes [6] (Figures 1 and 2).  It is assumed that lipophilic molecules are confined within the lipid bilayers whi the hydrophilic ones are retained in the niosomes' aqueous partitions. This efficient com partmentalization improves the stability of the enclosed drugs preventing their chemica and enzymatic degradation [7]. Proniosomes are nonionic dehydrated structured proves cles in the powdered form or in the gel states. Provesicles are water soluble dry free-flow ing granular products that can be immediately rehydrated before use avoiding many i sues related to aqueous vesicular dispersions. Proniosomes and niosomes can be pro duced by using cholesterol, non-ionic surfactants (Tween 20, 40, 80, Span20, 40, 60, 80, 85 solvents as chloroform and methyl and ethyl alcohols and lecithin. Usually, surfactan utilized to produce niosomes and proniosomes are characterized by low aqueous solubi ity but Tween can be successfully used to produce micelles on hydration [8].
Niosomes are similar to liposomes, but they are cheaper, exhibit a higher stability encapsulation efficiency, and permeability for small molecules, avoid the degradation o phospholipids by oxidation, and are easier to store and handle. Indeed, niosomes displa some drawbacks, such as aggregation, fusion, and leakage of drugs, while proniosome can overcome these issues contrasting leakage, aggregation, or hydrolysis of drugs whi optimizing their storage and biodistribution, adding the possibility of sterilization, room temperature storage, and being rehydrated instantly to create niosomes [9].
Proniosomes have several pluses over niosomes, contrasting leakage, aggregation, o hydrolysis of drugs while optimizing their storage and biodistribution. It is assumed that lipophilic molecules are confined within the lipid bilayers while the hydrophilic ones are retained in the niosomes' aqueous partitions. This efficient compartmentalization improves the stability of the enclosed drugs preventing their chemical and enzymatic degradation [7]. Proniosomes are nonionic dehydrated structured provesicles in the powdered form or in the gel states. Provesicles are water soluble dry free-flowing granular products that can be immediately rehydrated before use avoiding many issues related to aqueous vesicular dispersions. Proniosomes and niosomes can be produced by using cholesterol, non-ionic surfactants (Tween 20, 40, 80, Span 20, 40, 60, 80, 85), solvents as chloroform and methyl and ethyl alcohols and lecithin. Usually, surfactants utilized to produce niosomes and proniosomes are characterized by low aqueous solubility but Tween can be successfully used to produce micelles on hydration [8].
Niosomes are similar to liposomes, but they are cheaper, exhibit a higher stability, encapsulation efficiency, and permeability for small molecules, avoid the degradation of phospholipids by oxidation, and are easier to store and handle. Indeed, niosomes display some drawbacks, such as aggregation, fusion, and leakage of drugs, while proniosomes can overcome these issues contrasting leakage, aggregation, or hydrolysis of drugs while optimizing their storage and biodistribution, adding the possibility of sterilization, room temperature storage, and being rehydrated instantly to create niosomes [9].
Proniosomes have several pluses over niosomes, contrasting leakage, aggregation, or hydrolysis of drugs while optimizing their storage and biodistribution.

Composition Cargo Application Reference
Cholesterol and sorbitan monostearate Tamoxifen citrate Incorporated in hydrogel as a pH-responsive drug delivery for breast cancer treatment [75] Cholesterol and Span 20 Tamoxifen citrate and doxorubicin Breast cancer treatment [76] Cholesterol, Span60, PEG and TAT peptide Tenofovir HIV treatment [77] Cholesterol, Span 60 and Tween 40 Timolol maleate Glaucoma treatment [78] Cholesterol and Span 60 [79] Cholesterol  Magnetic properties and cargo-targeted delivery [107] Thanks to their capability to store and deliver both hydrophilic and hydrophobic medications through topical, oral, transmucosal, pulmonary, ocular, and parenteral/intravenous administration, niosomes and proniosomes are increasingly used as vaccines and treatments for infection, inflammation, cancer, and many other acute or chronic diseases.

Ethosomes
Ethosomes were designed and developed in 2000 by Touitou et al. [108] as an advanced noninvasive passive lipid-based delivery system. As represented in Figure 3, these carriers are lipid bilayers composed of phospholipids, water, and high concentrations of ethanol which gives them remarkable transdermal permeability skills. Ethanol and lipid molecules act in the polar head group region increasing membrane fluidity and permeability. Ethosomes have significantly improved skin delivery, carrying the active compounds in the deeper layers of the skin in occlusive and non-occlusive conditions. In addition, they display high deformability, encapsulation efficiency, stability, biocompatibility, and a negative charge due to ethanol that leads to small vesicles size, enhancing the bioavailability of the compounds. Despite these advantages, there are some drawbacks caused by the volatile nature of ethanol, such as problems related to system instability, drug leakage, and skin irritation [109]. These vesicles are successfully used for topical administration of a considerable variety of drugs such as antifungals, antivirals, antibiotics, anti-inflammatories, and many others as detailed in Table 3

Ethosomes
Ethosomes were designed and developed in 2000 by Touitou et al. vanced noninvasive passive lipid-based delivery system. As represented in carriers are lipid bilayers composed of phospholipids, water, and high co ethanol which gives them remarkable transdermal permeability skills. Eth molecules act in the polar head group region increasing membrane fluidit bility. Ethosomes have significantly improved skin delivery, carrying t pounds in the deeper layers of the skin in occlusive and non-occlusive con dition, they display high deformability, encapsulation efficiency, stability, ity, and a negative charge due to ethanol that leads to small vesicles size, bioavailability of the compounds. Despite these advantages, there are so caused by the volatile nature of ethanol, such as problems related to sys drug leakage, and skin irritation [109]. These vesicles are successfully used ministration of a considerable variety of drugs such as antifungals, antivir anti-inflammatories, and many others as detailed in Table 3.

Composition
Cargo Application 5-Aminolevulinic Treatment of

Transfersomes
Many drug delivery systems have been designed over the past decades for transdermal administration, which offers many advantages over other routes thanks to its capability of escaping presystemic metabolism, tune drug release reducing variation in drug levels, enhancing pharmacological response. Compared to most other transdermal delivery methods including chemical permeation enhancers, sonophoresis, microneedles, lipid vesicles thanks to their distinctive composition can transport both hydrophilic and lipophilic drugs [140].
Among the LNV, transfersomes, first proposed in the early 1990s, are ultra-deformable elastic vesicles successfully employed as a non-occluded method able to permeate skin through the stratum corneum reaching the dermis and blood circulation [141]. As schematized in Figure 4, they are firstly characterized by an aqueous core enclosed by a lipid bilayer of amphipathic constituent as phosphatidylcholine, lecithin, or a mixture of lipids. In addition to a very low percentage of alcohol (3-10%), they are made with 10-25% of bilayer-softening complexes, surfactants, or edge activators as Tweens, Spans, sodium cholates, and deoxycholate. The appropriate phospholipids/surfactants ratio tunes transferosomes' membrane elasticity reducing vesicles' rupture chances through the skin [142,143]. By having edge activators in their structure, thanks to their remarkable elastic properties, transfersomes defeat many main liposomes' weaknesses resulting in more apt to squeeze themselves through the skin barrier [144]. Despite these advantageous properties, transfersomes exhibit also some drawbacks, i.e., chemical instability due to the oxidative degradation and expensiveness in the precursors and manufacturing [143]. Thanks to their enhanced skin-penetrat set up skin drug storage area for continuous t as well as high, molecular weight drugs as Thanks to their enhanced skin-penetration abilities, transfersomes are competent to set up skin drug storage area for continuous therapeutic molecules delivery releasing low, as well as high, molecular weight drugs as antioxidants, chemotherapy, anti-Inflammatory, and corticosteroids (Table 4). Phospholipon 90 G ® and sodium cholate Pentoxifylline Treatment of intermittent claudication and chronic occlusive arterial diseases [157] Lecithin and Tween 20/80 Resveratrol Antioxidant [158] Soy phosphatidylcholine, Tween 80 and ceramide III Retinyl palmitate Antioxidant [159] Soy phosphatidylcholine and emu oil Tamoxifen Transdermal therapy for breast cancer [160] Soy lecithin and Tween 80 Taxifolin Antioxidant [161] Soy phosphatidylcholine and Tween 80 Tocopherol Antioxidant [162] Soya lecithin and Tween 80 Zolmitriptan Migraine treatment [163] Soy lecithin and sodium deoxycholate Human growth hormone Transdermal hormone delivery [164] Egg phosphatidylcholine, stearylamine and Tween 20 PnPP-19 peptide Treatment of erectile dysfunction [165]

Pharmacosomes
The name pharmacosomes refers to the amphiphilic, zwitterionic, stoichiometric complexes of polyphenolic compounds with phospholipids, as schematized in Figure 5. The success in the use of pharmacosomes is explained by the surface and bulk interactions of lipids with drugs since the latter possess an active hydrogen atom as -OH, -COOH, -NH 2 , which can be esterified to the lipid causing an amphiphilic compound [166,167].
The use of pharmacosomes in drug delivery has several advantages over that of other vesicles such as niosomes, transferosomes, and liposomes. More in detail, any active molecules in which a carboxyl group is present can be esterified without a spacer chain as opposed to those characterized by the presence of amino or hydroxyl groups which, in order to be esterified, require spacer groups. Pharmacosomes design is based on the phospholipids/water superficial and bulk interaction; the drug molecule and the connected lipid molecule, respectively, behave like the polar head group and the lipidic chain giving the molecule an amphipathic character. Thanks to their hydrophilic and lipophilic properties, these lipid LNV improve drugs' dissolution in gastrointestinal fluid, increasing the bioavailability of low soluble treatments avoiding leak and rupture release [168,169]. Pharmacosomes' in vivo pharmacokinetic performances are conditioned by vesicles' dimension, by the drug molecule's functional groups, by the lipids' fatty acid chain length, and, last but not least, by the spacer groups' availability. The high tunability of each of the components listed above makes these types of vesicles excellent candidates for the effective delivery of a wide range of active molecules including anti-cancer and anti-inflammatory remedies (Table 5) [170].

Pharmacosomes
The name pharmacosomes refers to the amphiphilic, zwi complexes of polyphenolic compounds with phospholipids, as s The success in the use of pharmacosomes is explained by the surfa of lipids with drugs since the latter possess an active hydrogen a NH2, which can be esterified to the lipid causing an amphiphilic c The use of pharmacosomes in drug delivery has several adva vesicles such as niosomes, transferosomes, and liposomes. More i ecules in which a carboxyl group is present can be esterified wi opposed to those characterized by the presence of amino or hyd order to be esterified, require spacer groups. Pharmacosomes desi pholipids/water superficial and bulk interaction; the drug mole lipid molecule, respectively, behave like the polar head group and Among the few limitations relating to the use of pharmacoses, reference should be made to their susceptibility to hydrolyzation, fusion, or aggregation during storage or engineering processes [171,172]. Table 5. Pharmacosomes' drug delivery applications.

Ufasomes
Unsaturated fatty acid vesicles preparation, more commonly known as ufasomes, was first reported in 1973 by Gebicki and Hicks [180]. In a controlled pH range, from 7 to 9, they are a closed lipid bilayered suspension, made from unsaturated fats and their ionized species. In detail, fatty acid molecules' hydrocarbon tails are directed toward the deeper membrane layer while the carboxyl heads are in contact with water [181], as schematized in Figure 6. Oleic and linoleic acid (cis, is-9,12-octadecadienoic acid), the major ufasomes' constituents, confer to these nanovesicles a more versatile nature than that of the other LNV, by ranking them between different nanosystems formed from double-chain amphiphiles and from single-chain surfactants micelles. Their biochemical composition makes them easily to assemble and real biocompatible [182,183]. By enhancing ufasomes stability with the identification of the appropriate fatty acid, pH range, and lipoxygenase amount, increasingly targeted and effective drug delivery solutions are being developed (Table 6).

Phytosomes
Although for a long time phyto-pharmaceuticals have a promin therapeutic scene, it should be emphasized how phyto-active constit flavonoid, and terpenoids demonstrate considerable in-vitro bio-actio

Composition Cargo Application Reference
Cholesterol and oleic acid Cinnarizine Antihistaminic activity [184] Phosphatidylcholine from soy and oleic acid

Minoxidil
Hypertension treatment [185] Phosphatidylcholine from soy oleic and linoleic acid

Phytosomes
Although for a long time phyto-pharmaceuticals have a prominent position in the therapeutic scene, it should be emphasized how phyto-active constituents as phenolics, flavonoid, and terpenoids demonstrate considerable in-vitro bio-action but are still characterized by low in-vivo effectiveness due to their high molecular weight, low lipid solubility, and bioavailability [188]. Phytosomes nanovesicles originating by Phyto-Phospholipid Complex (PPC), have been developed as a capable strategy to improve natural drugs delivery and bioavailability. PPCs originate by the phospholipids' polar head and active constituents' interactions. The two long fatty acid chains do not take part in the formation of the complex, they can interchange encapsulating the polar region of complexes originating a lipophilic side when resuspended in water (Figure 7) [189].
lipid Complex (PPC), have been developed as a capable strategy to im delivery and bioavailability. PPCs originate by the phospholipids' po constituents' interactions. The two long fatty acid chains do not take p of the complex, they can interchange encapsulating the polar region nating a lipophilic side when resuspended in water (Figure 7) [189]. Phytosomes have many structural and functional aspects in common with liposomes and tranferosomes such as the capability to improve the solubility of weakly soluble polyphenolic phytochemicals. Otherwise, phytosomes and transferosomes are more stable than liposomes in 4 • C and 25 • C aqueous media up to three months since liposomes should be freeze dried to preserve their stability. Phytosomes, as well as transferosomes, exhibit superior dermal penetration properties leading noticeable accumulation in the epidermis and dermis. Since the phytosomes configuration is grounded on the H-bond interaction between the phospholipid molecules' polar moiety and the phytoconstituents, the laded compounds permanence is higher than in other lipid nanovesicles [190]. The numerous and very recent drug delivery applications collected in Table 7 show how phytosome nanotechnology will definitely get more efficient the ways of bioactive phytochemicals therapeutic and aesthetic delivery counteracting the bottlenecks of the low absorption and poor penetration rate across biological barriers improving herbal-originated compounds pharmacodynamic and pharmacokinetic and assets [190]. Table 7. Phytosomes' drug delivery applications.

Catanionic Vesicles
An innovative class of biocompatible and biodegradable drugs lipidic nanovehicle is represented by the catanionic vesicles for their capability to improve the stability and cellular uptake of a wide range of active molecules [215]. These hybrid nanovesicles spontaneously form when unequal amounts of cationic and anionic single-tailed surfactants are dispersed in water [216] (Figure 8).

DPPC Rutin
Antioxidant for the prevention of liver inflammation

Catanionic Vesicles
An innovative class of biocompatible and biodegradable drugs lipidi represented by the catanionic vesicles for their capability to improve the s lular uptake of a wide range of active molecules [215]. These hybrid nano neously form when unequal amounts of cationic and anionic single-tailed dispersed in water [216] (Figure 8). These nanovesicles are produced by using easily accessible cheap sur comparison with phospholipid vesicles, are thermodynamically advanta colloidal stability. Alkyl ammonium bromide and gemini surfactants suc nary ammonium salts have been used for catanionic vesicles production; they are cytotoxic and not biodegradable, the conjugation with safer mo successfully considered [217]. Their low production costs, higher stability ing capability, together with the fact that they suffer less from ruptures and These nanovesicles are produced by using easily accessible cheap surfactants and, in comparison with phospholipid vesicles, are thermodynamically advantaged in terms of colloidal stability. Alkyl ammonium bromide and gemini surfactants such as bis-quaternary ammonium salts have been used for catanionic vesicles production; however, since they are cytotoxic and not biodegradable, the conjugation with safer molecules is being successfully considered [217]. Their low production costs, higher stability and drug loading capability, together with the fact that they suffer less from ruptures and pressure drops make them excellent drug delivery vehicles for vaccination and anti-microbial, cancer, and inflammatory applications (Table 8). Thus, although catanionic vesicles have a huge applicability in biomedicine, they can suffer safety problems due to their eventual low bio-and emocompatibility. Numerous ongoing researches point to the optimization of their morphology, hydrophobicity, and ionic charge by carefully choosing the proper surfactant and by tuning the anionic/cationic surfactant ratio eventually adding some suited additive [218].  Antimicrobial activity [238] C: NαNω-Bis(Nαcaproylarginine) α,ω-propyldiamide A: Lichenysin Antimicrobial and antifungal activity [239] C: N(π), N(τ)-bis(methyl)-L-Histidine tetradecyl amide A: lysine-based surfactant Nα-lauroyl-Nεacetyl lysine or sodium myristate Antimicrobial activity [240] C: N-dodecylamino-1-deoxylactitol A: ketoprofen Anti-inflammatory activity [241]

Extracellular Vesicles
The most heterogeneous and versatile class of lipid vesicles is certainly that of extracellular vesicles (EVs) (Figure 9) including apoptotic bodies, microvesicles, and exosomes. These vesicles are ubiquitarian and can be isolated from cells culture media and from all the major biological fluid as urine, plasma, saliva, amniotic and cerebrospinal fluid, semen, among others [242][243][244][245]. Both apoptotic bodies and microvesicles, with dimensions ranging between 500 nm and 2 µm and from 50 nm to 1 µm, respectively, arise from plasma cell membrane outward blebbing and fragmentation. On the other side, exosomes, deriving from the endocytic pathway, have diameters between 30 to 120 nm [246]. Many authors reported about the EVs use in drug delivery since their surface is characterized by antigens, related to the parental cells, able to direct specific homing or targeting phenomena [247]. Although the EVS, as the main physio-pathological intracellular communication mediators, are already in origin able to transport miRNA, proteins, and other biological molecules, their morpho-functional and biochemical characteristics make them excellent candidates for post isolation nanotechnological modifications. In the last twenty years, numerous studies show the great potential of these vesicles in both the diagnostic and therapeutic fields [248]. Their high biocompatibility, low immunogenicity coupled with a superior loading capability make them proper tools for post isolation drug delivery load and engineering. In addition to a whole series of chemical or biological functionalization, many studies are referring to the possibility of loading them with cellular organelles such as mitochondria, NPs, drugs, and nuclei acids [249][250][251]. Although the intrinsic complexity related to the EVs' size and natural (batch-tobatch) heterogeneity makes their drug delivery application much more complex than that with merely synthetic production systems, many exogenous EVs' active molecules loading methods have been successfully proposed for the clinical EVs' translation [252] (Table Although the intrinsic complexity related to the EVs' size and natural (batch-to-batch) heterogeneity makes their drug delivery application much more complex than that with merely synthetic production systems, many exogenous EVs' active molecules loading methods have been successfully proposed for the clinical EVs' translation [252] (Table 9). Table 9. Extracellular vesicles' drug delivery applications.

Parental cell Cargo Application Reference
EVs from HEK293T cells Angiotensin converting enzyme II (ACE2) Protect from SARS-CoV-2 infection by competitively bound to virus against host cells [253] Milk-derived exosomes Anthocyanidins Anti-proliferative and anti-inflammatory in lung cancer [254] Exosomes from breast and colorectal cancer cells Aspirin Cancer therapy [255] Exosomes from MIN-6 cells BAY55-9837 Increase insulin production for type 2 diabetes mellitus [256] Exosomes from macrophages Berberine Spinal cord injury treatment [257] EVs from human umbilical cord mesenchymal stem cells Cannabidiol Increase the therapeutic efficacy of doxorubicin in triple negative breast cancer [258] Exosomes from umbilical cord-derived macrophages Cisplatin Ovarian cancer cells treatment [259] EVs from macrophages Curcumin Neuroprotection and ischemia-reperfusion injury treatment [260] Inhibit the phosphorylation of Tau protein [261] Exosomes from mesenchymal stem cells Attenuate the progression of osteoarthritis [262] EVs from HEK293 cells Myocardial infarction treatment [263] Exosomes from bone marrow-derived mesenchymal stem cells Cerebral ischemia treatment [264] Exosomes from HEK293 cells Curcumin and RAGE-binding peptide Acute lung injury treatment [265] EVs from smooth muscle cells Cystatin C Protection and healing of the nervous system in different neurotoxic conditions [266] Exosomes from lung cancer Docetaxel Non-small cell lung cancer treatment [267] Exosomes from cervical cancer Cervical cancer treatment [268] Exosomes from blood samples Dopamine Parkinson's disease treatment [269] EVs from macrophages Doxorubicin Metastatic ovarian cancer treatment [270] Exosomes from mesenchymal stem cells Colorectal cancer treatment [271] Exosomes from human glioma Glioma treatment [272] Milk-derived exosomes Cancer treatment [273] Exosomes from HEK293 cells Cancer treatment [274] Exosomes from bone marrow-derived mesenchymal stem cells Osteosarcoma treatment [275] Exosomes from colon cancer Colorectal cancer treatment [276] Exosomes from human breast and ovarian cancer Breast and ovarian cancer treatment [277] Exosomes from macrophages Edaravone Permanent middle cerebral artery occlusion treatment [278] Exosomes from human fetal lung fibroblasts Erastin Triple-negative breast cancer therapy [279] Exosomes from pancreatic cells Gemcitabine Pancreatic cancer treatment [280] EVs from human plasma Imperialine Non-small cell lung cancer treatment [281] EVs from human umbilical vascular endothelial cells

Parental cell Cargo Application Reference
Exosomes from embryonic stem cells Paclitaxel Glioblastoma treatment [285] Exosomes from mesenchymal stem cells Carcinoma treatment [286] EVs from gingival mesenchymal stromal cells Cancer treatment [287,288] Exosomes from macrophages Pulmonary metastases treatment [289] Milk-derived exosomes Lung cancer treatment [290] EVs from bone marrow mesenchymal stromal cells Malignant pleural mesothelioma treatment [291] Exosomes from macrophages Multiple drug-resistant cancer treatment [292] EVs from lung cancer cells Paclitaxel and oncolytic virus Primary and metastatic cancer treatment [293] EVs from neutrophil-like cells Piceatannol Alleviated acute lung inflammation/injury and sepsis induced by lipopolysaccharide [294] Exosomes from plasma Quercetin Relieve symptoms of Alzheimer's disease by inhibiting phosphorylation of Tau and reducing the formation of insoluble neurofibrillary tangles [295] Exosomes from human ovarian cancer Triptolide Ovarian cancer treatment [296] Mannosylated exosomes from macrophages Vancomycin and lysostaphin Eradication of intracellular quiescent MRSA [297] Exosomes from fibroblasts WNT3A Repair of osteochondral defects [298] Many types of cell-derived exosomes, coming from both plant and human eukaryotic cells, have recently been used to successfully encapsulate inorganic NPs. The cargo can be either loaded by treating parental cells or by post EVs isolation engineering [299]. The potential benefits of a wide range of inorganic NPs-loaded EVs have been proven in various drug delivery applications as extensively listed in Table 10. Grapefruit EVs Doxorubicin-loaded heparin-based NPs Glioma treatment [301] Exosomes from melanoma cells

Gold NPs
Cancer treatment [302] Exosomes from HEK293T cells Blood-brain barrier penetration and brain disorders future treatments [303] Exosomes from bone marrow mesenchymal stromal cells Neuroimaging for various brain disorders [304] Exosomes from mesenchymal stem cells [305] Exosomes from breast cancer cells Gold iron oxide hybrid NPs MRI contrast agent and photodynamic therapy [306] Exosomes from mesenchymal stem cells Iron oxide NPs Myocardial infarction treatment [307] Wound repair [308] Increase activation and migration ability of macrophage [309] Tumor cell ablation via magnetically induced hyperthermia [310] EVs from human umbilical vascular endothelial cells Photodynamic and hyperthermia therapy of prostate cancer [311] Exosomes from macrophages Laurate-functionalized Pt(IV) prodrug, human serum albumin, and lecithin NPs Breast cancer and metastatic breast cancer lung nodules treatment [312]

Parental Cell Cargo Application Reference
Exosomes from lung adenocarcinoma cells

Metal-organic framework
Detection of the ATP level in living cancer cells, providing an efficient tool for the cell metabolism study [313] Exosomes from triple negative breast cancer cells Delivery of anticancer compounds [314] Exosomes from HeLa cells [315] Exosomes from lung cancer or glioma Palladium nanosheet Deliver catalytic cargo directly to cancer cells [316] Exosomes from triple negative breast cancer cells

PLGA NPs
Cancer therapy [317] Exosomes from lung carcinoma cells [318] EVs from Staphylococcus aureus Intracellular delivery of antibiotics for intracellular pathogen-associated complications treatment [319] Exosomes from breast cancer Quantum dots of vanadium carbide Cancer photothermal therapy [320] Exosomes from hepatocellular carcinoma Silver and iron NCs Cancer bioimaging [321] Exosomes from macrophages SPIONs and curcumin Synergistic antitumor therapy in gliomas [322] Exosomes from plasma Superparamagnetic magnetite colloidal nanocrystal clusters Cancer treatment [323] EVs from KB cells Zinc oxide NCs Cancer treatment [324] Since EVs are remarkably involved in genetic information transfer in normal and pathological states [325][326][327], it is not difficult to see their potential as engineered nucleic acids carriers for drug the treatment of ischemic stroke, myocardial infarction [328], traumatic brain injuries [329], and liver fibrosis [330].
The intrinsic properties of EVs such as low immunogenicity and safety make them a suitable candidate for gene cancer therapy with promising advantages with respect to the conventional chemotherapeutic treatments. EVs transfer their RNA or DNA cargo to the target cells with the aim to alter the tumoral genes information and act, e.g., as tumoral suppressors. In addition, the therapeutic properties of EVs-nucleic acids loaded can be further improved by tailoring their surface [331] in order to maximize specificity and successful delivery. In Massaro et al. [332] is reported a list of the ligands used for cancer therapy. Interestingly, attempts to conjugate RNAs to molecules such as cholesterol for EVs surface functionalization were reported [333,334], with the aim to improve loading control and delivery. Therapeutics effects of Plasmid DNA, mRNA, miRNA, and shRNA delivery EV-mediated were reported in Table 11 underlining how gene therapy combined with EVs delivery is a rapidly growing field for safe and effective precision medicine treatments. Table 11. Extracellular vesicles' nucleic acids delivery applications.

Parental Cell Cargo Application Reference
Microvesicles from breast cancer cells Minicircle DNA encoding a thymidine kinase /nitroreductase fusion protein Breast cancer therapy [335] EVs from mice melanoma cells Plasmid DNA coding for ESAT-6 Promote antitumor activity of dendritic cells [336] EVs from human brain endothelial cells and macrophages Plasmid DNA encoding for brain-derived neurotrophic factor Protection of the brain endothelium increasing endothelial ATP levels [337] EVs from macrophage cells Tripeptidyl peptidase-1-encoding plasmid DNA Lysosomal storage disorder, Neuronal Ceroid Lipofuscinoses 2 (CLN2) or Batten disease treatment [338] EVs from red blood cells Anti-miR-125b ASOs and Cas9 mRNA Cancer treatment [339] Exosomes from mouse neuronal cells miR-21-5p Suppression of autophagy after a traumatic brain injury [340] Table 11. Cont.

Parental Cell Cargo Application Reference
EVs from HEK-293T cells HChrR6 mRNA Convert CNOB into MCHB for the treatment of cancers [367] Exosomes from mesenchymal stem cells, dendritic cells or HEK-293T cells PTEN mRNA Restore tumor-suppressor function in PTEN deficient gliomas [368] EVs from non-pigmented ciliary epithelium cells anti-fibrotic (SMAD7) siRNA Lower intraocular pressure in primary open-angle glaucoma [369] Exosomes from autologous breast cancer cells Cationic bovine serum albumin conjugated siS100A4 Suppress postoperative breast cancer metastasis [370] EVs from murine neuroblastoma cell line and dendritic cells Cholesterol-conjugated siRNAs Human antigen R silencing for cancer treatment [334] Exosomes from HEK-293T cells c-Met siRNA Reverse chemoresistance to cisplatin in gastric cancer [371] Exosomes from HEK-293T cells Hepatocyte growth factor (HGF) siRNA Inhibitory effect on tumor growth and angiogenesis in gastric cancer [372] EVs from mesenchymal stem cells derived from umbilical cord Wharton's jelly Hydrophobically modified asymmetric siRNAs conjugated with cholesterol Huntingtin silencing in neurons [333] Exosomes from glioblastoma cells [373] Exosomes from human neuroblastoma cells Heat shock protein-27 (HSP27) siRNA Decrease of cell differentiation toward mature neuron in neuroblastoma [374] Exosomes from urine-derived induced pluripotent stem cells ICAM-1 siRNA Alleviating inflammation of pulmonary microvascular endothelial cells [375] Exosomes from HEK-293T cells KRAS siRNA Inhibition of tumor growth [376] EVs from astrocytes LincRNA-Cox2 siRNA Lipopolysaccharideinduced microglial proliferation for treatment of CNS disorders [377] Exosomes from mesenchymal stem cells PTEN siRNA Promote recovery for spinal cord injury individuals [378] EVs from red blood cells P65 and Snai1 siRNA Inhibit renal inflammation and fibrosis for acute kidney injury treatment [379] EVs from HEK-293T cells RAGE siRNA Attenuated inflammation in myocarditis [380] Exosomes from bone-marrow-derived mesenchymal stem cells siGRP78 Suppress Sorafenib resistance in hepatocellular carcinoma [381] Exosomes from bovine milk siKRAS Lung tumor treatment [382] EVs from different cell lines siRNA Reducing the therapeutic dose of siRNA for different pathologies [383] EVs from human umbilical cord mesenchymal stem cells siRNA-ELFN1-AS1 Inhibit colon adenocarcinoma cells proliferation [384] Exosomes from normal human foreskin fibroblast siRNA or short hairpin RNA specific to oncogenic Kras G12D Pancreatic ductal adenocarcinoma treatment [385] Exosomes from HEK-293T cells Transient receptor potential polycystic 2 (TRPP2) siRNA Reduce the epithelial-mesenchymal transition in pharyngeal squamous carcinoma [386] Exosomes from brain endothelial bEND.3 cells Vascular endothelial growth factor (VEGF) siRNA Knockdown of VEGF in brain cancer cells [387] Exosomes from HEK-293T cells Different viral products including Ebola Virus VP24, VP40 and NP, Influenza Virus NP, Crimean-Congo Hemorrhagic Fever NP, West Nile Virus NS3, and Hepatitis C Virus NS3 Exosomes-based vaccines [388]

Conclusions
It is well known that liposomes, assumed to be the oldest category of lipidic nanovesicles, have been broadly considered as the major candidates for biomedical and drug de-livery applications. Despite their high biocompatibility and the ability to effectively carry both hydrophilic and/or hydrophobic active molecules to the target site, they still suffer some unresolved weaknesses such as brief shelf-life, low colloidal stability, and limited and expensive preparation methods [389]. The development of new drug delivery approaches has significantly boosted the design and the production of the just reviewed non-liposomal lipid nanovesicles. This new cohort of lipid vesicles can complement liposomes as alternative nanovesicular drug delivery systems and although recently implemented, they have all the chances to overspread as successful engineered nanomaterials.
Considering the existent non-liposomal LNV, those collected in this review, given their countless listed applications, have undoubtedly proved to be the most successful ones by reaching clinical use. Surely among the different types of LNV described in this review, those of cellular origin, the extracellular vesicles, are those that could also give future results closer to the needs of personalized medicine therapeutic plans. The possibility of isolating them from the same patient who is going to be treated reduces the likelihood of rejection phenomena both by increasing the compliance of the therapy and by reducing any adverse effects. Therefore, it would be foreseen that very soon, the LNV carrier's production will scale-up from the lab scale to the industrial one issuing high-quality competitive outcomes.
In this regard, we would like to conclude with an update on the recent and promising use of lipid nanovesicles for the nucleic acids based-vaccine development. This application has been mainly oriented to the oncologic field, but recently, under the pressure of the latest terrible health emergency that has afflicted the entire globe, anti-viral applications have been reported. EV-based vaccines to deliver mRNA coding for specific molecules such as proteins or by the exposure of specific features on EVs surface have been designed. Since 2020, the SARS-CoV-2 pandemic has boosted additional efforts for the successful design of forceful vaccines [332,390]. Leading approved vaccines provide immunization by the viral Spike (S) protein, injected as purified proteins or codified by the administered mRNAs sequences and showing that "mRNA-based vaccines can fill the gap between emerging pandemic infectious disease and a bountiful supply of effective vaccines" [391]. The mRNA-based vaccine BNT162b2 was developed by Pfizer/BioNTech while the mRNA-1273 SARS-CoV-2 vaccine was developed by Moderna [392]. In Tsai et al. [364] was reported another approach for SARS-CoV-2 vaccines: exosomes are used to deliver mRNAs sequences with the aim to express not only the spike protein but also another artificial protein named "LSNME" and containing the viral spike, nucleocapsid, membrane, and envelope proteins. This approach has been tested on mice with promising results and, along with the many other applications reported in this review, confirmed the growing potential of lipid nanovesicles-mediated delivery as an effective tool for the translation of nanotechnology, bioengineering, and nanomaterials studies from research to clinic.