Lipid-based NPs

Lipid-based NPs are the oldest and broadest group of NCs that have been used in drug and gene delivery. The milestone that decided to start research on liposomal NC was the approval in 1995 of the drug Doxil, a liposomal preparation of DOX for second-line therapy of Kaposi’s sarcoma. Doxil therapy was associated with a lower percentage of adverse effects, such as hair loss or peripheral neuropathy, and most importantly, with lower cardiotoxicity while maintaining high treatment efficacy [44]. The positive conclusion of research on the first type of NP encouraged researchers to continue working in this direction. Over the past 30 years, intensive research has led to the approval of 23 products in the US and Europe, 63% of which were for cancer therapy [45].

Five generations of lipid NCs can be distinguished: liposomes, solid lipid NPs (SLNs), nanostructured lipid carriers (NLCs), lipid drug conjugates (LDCs), and hybrid polymer-lipid NPs (PLNs).

Liposomes are composed of a lipid bilayer enclosing a hydrophilic core inside the NC. Due to this structure, they are ideal carriers because hydrophilic drugs can usually be placed in the core, while hydrophobic drugs are trapped in the lipid bilayer. Individual liposomal NCs differ in size, lamellarity (number of lipid layers), and surface charge. They can be classified as single- or multilayer, while single-layer liposomes can be further divided into small [small unilamellar vesicles (SUVs); 70 nm] and large [large unilamellar vesicles (LUVs); 100–250 nm]. Both SUVs and LUVs consist of a single lipid bilayer and a large aqueous core; hence, they are mainly suitable for transporting hydrophilic drugs, whereas multilayer liposomes, composed of several lipid bilayers and a limited aqueous space, are used for transporting hydrophobic drugs [46]. Furthermore, based on the charge of the lipid components, cationic and anionic liposomes are distinguished. Cationic liposomes have been used for transporting nucleic acids containing negatively charged phosphate groups; therefore, they can serve as vectors for miRNA, DNA, and siRNA therapies [47]. In turn, anionic liposomes have been shown to be effective in transporting plasmid DNA [48]. An example of a liposomal drug carrier is the previously mentioned form of Doxil, in which the drug is located in the aqueous phase of the core as a crystalline salt (DOX)₂SO₄, which is transported into the interior of the liposome due to the transmembrane gradient of ammonium ions, which also determines the release of the drug in the tumor [49].

Liposomes are not ideal carriers; their main disadvantages are the relatively expensive preparation process, a relatively low degree of drug load capacity and stability, most importantly, rapid decomposition in the human body before the drug reaches therapeutic concentration [50]. Due to these limitations, research has been conducted to introduce new, improved generations of lipid NCs.

SLNs consist of fully crystallized lipid components, characterized by a highly ordered crystalline structure in combination with emulsifiers. Their advantages include: high stability, excellent drug protection, and their controlled release, which drew the attention of researchers as early as 1991 [51]. However, they have two main disadvantages, which are poor, long-term drug retention and low drug loading capacity. This is due to the storage-related change in the structure of the lipid matrix to a more ordered one with simultaneous gradual excretion of encapsulated drugs [52]. Due to these limitations, solutions were sought to improve the stability of this form of NC. By incorporating liquid lipids into their structure, NLCs have gained an advantage over SLNs. The mixture of solid and liquid lipids takes the form of a partially crystallized lipid structure, which provides better stability and greater drug loading capacity with minimal leakage during storage [53]. Initially, they were considered for the transport of lipophilic drugs, but over time, it turned out that they can also effectively transport hydrophilic drug molecules. Studies comparing the efficacy of co-delivery of vincristine (VCR) and temozolomide (TMZ) using engineered lipid molecules, SLNs and NLCs showed significantly higher anticancer efficacy against glioma cells for drugs transported using NLCs compared to SLNs, both in vitro and in vivo [54]. LDCs are particularly well suited for the delivery of water-soluble drugs, due to the ability of the LDC lipid matrix to conjugate with hydrophilic drug molecules (via covalent bonding or salt formation with fatty acids). They are considered the best method to increase the bioavailability of drugs with limited absorption during oral administration due to the increased ability of the NC to avoid enzymatic degradation or damage due to changing pH values of the gastrointestinal environment [55]. A study on the efficacy of LDC NPs for the delivery of 5-fluorouracil (5-FU) to brain tumor cells confirmed the higher cytotoxicity of the NC-conjugated form of the drug against glioma cells. It was shown that LDC provides higher drug concentrations in the systemic circulation (blood), while pure 5-FU was largely available in peripheral tissues. The half-life of the conjugated form of the drug was approximately 12 times longer, and the maximum concentration in brain tissue was twice as high as in the traditional form of the drug, which clearly proved the greater efficacy of glioma therapy using LDC [56]. Hybrid PLNs consist of at least two types of materials integrated into one drug delivery system, combining the advantages of each of the individual components. Classic PLNs have a core-shell structure (polymer core surrounded by a lipid layer) and are derived from both liposomes and polymer NPs [57]. Due to this structure, they are characterized by high stability and high biocompatibility. Due to the dual structure, this method is particularly interesting in terms of the possibility of combined drug delivery, which is particularly useful in oncology. An example is a loaded lipid-polymer hybrid NP containing DOX and edelfosine (EDL), which was designed to increase the efficacy of osteosarcoma treatment. It was shown that the targeted NPs were characterized by increased cellular internalization and subcellular distribution in cancer cells. Additionally, it was proven that the combination of drugs in a 1:1 ratio achieves the most synergistic effect in inducing cancer cell death in in vitro studies. In vivo results confirmed high efficacy in destroying cancer cells without detectable side effects. These results indicate that hybrid lipid-polymer NCs, due to their properties such as better physiological stability, the ability to carry a large drug load, drug co-delivery, and prolonged blood circulation, are promising in terms of increasing the efficacy of oncological treatment [58].

The figure below (Figure 3) shows the structural diagrams of selected forms of lipid-based NPs.

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Figure 3. Schematic diagram of the structure of selected forms of lipid-based nanoparticles (LNPs). LNPs are diverse in structure but typically consist of a lipid core and a shell, often containing phospholipids, cholesterol, and PEGylated lipids. They can be categorized into liposomes, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and hybrid polymer-lipid nanoparticles (PLNs)

In addition to lipid-polymer hybrid NPs, lipid-metal hybrid NPs have also been designed, such as lipid-coated silver hybrid NPs (lipid-AgNPs), lipid-aluminum NPs, and liposome gold NPs (LiposAuNPs) [59, 60]. Metal-containing NPs are widely used for probing and labeling, but in vivo studies on their use for drug transport have shown some limitations due to low cellular uptake and poor stability. These assumptions have been confirmed, among others, in studies on AuNPs coated with lysophosphocholine. The results showed that lysophosphocholines-AuNPs formed aggregates with a core-shell structure, characterized by high stability, without significant structural changes, even when the temperature was increased to 50°C. Additionally, AuNPs showed the ability to kill cancer cells using photothermal therapy. In vivo studies in a mouse tumor xenograft model showed complete ablation of the tumor mass using near infra-red (NIR) laser illumination (750 nm), thus prolonging disease-free survival [61].

Block copolymer micelles

Block copolymer micelles are formed by the self-assembly of amphiphilic or oppositely charged copolymers in an aqueous environment. Hydrophilic and hydrophobic materials interact to form the corona and core of the micelle, respectively. They are particularly advantageous for poorly soluble, small molecules with high potency and significant toxicity, due to the possibility of encapsulating them inside the micelle. The appropriate size of the micelles determines the long circulation time. The micelles are large enough to avoid renal excretion (> 50 kDa), but small enough (< 200 kDa) to bypass filtration through the gaps between the endothelial cells in the spleen. In addition, it is believed that the presence of a non-ionic water-soluble coating and the appropriate size (10–100 nm) of polymer micelles limits their uptake by the mononuclear phagocyte system and allows passive penetration into the tumor tissue and retention of micelles in the tumor microenvironment. Currently, efforts are being made to increase the physical stability of micelles, which would increase the resistance to dissociation of this carrier and allow for higher drug accumulation within the tumor [62]. Micelles are a particularly good solution for the transport of substances with limited bioavailability. An example is quercetin (QUE)—a therapeutic substance with potential anticancer activity. The low bioavailability of QUE is a consequence of its lipophilic nature and low solubility in water, gastric juice, as well as poor permeability through the barrier in the form of the gastrointestinal epithelium, which results in low QUE absorption when administered orally. The use of micelles, in addition to increasing the bioavailability of drugs, can also be used to reduce the side effects resulting from the use of highly cytotoxic drugs, especially in oncological therapy. For example, DOX is one of the most potent anticancer drugs, but its use is limited due to strong cardiotoxicity resulting from the generation of free radicals and the presence of highly cardiotoxic metabolites, such as doxorubicinol. The toxic effect of this drug could be limited as a result of the use of strong antioxidants, such as QUE, which has the ability to scavenge free radicals and bind transition metal ions. In the study conducted by Chang et al. [63], lecithin-stabilized polymeric micelles (L_sbPMs) were prepared for the transport of QUE and the effect of combining QUE with DOX in micelles was examined. Cytotoxicity assay showed that QUE-loaded L_sbPM had significant antitumor activity against human breast cancer cells in vitro and in a mouse colon cancer model. In a pharmacokinetic study, compared with free QUE, intravenous and oral administration of QUE-loaded L_sbPM significantly increased the relative bioavailability to 158% and 360%, respectively, and the absolute bioavailability to 5.13%. The action of QUE-loaded L_sbPM combined with DOX could effectively inhibit the growth of colon cancer cells and reduce the toxicity to the heart tissue in a mouse model, which proved the effect of QUE on enhancing the antitumor activity and reducing the cardiotoxicity [63].

Albumin NCs

Albumins are ubiquitous, biodegradable, biocompatible, non-toxic, and non-immunogenic protein NCs. Albumin NPs can be easily modified by introducing functional groups on their surface or by enriching them with specific ligands directing the drug to a specific molecular target. Additionally, cancer cells have an increased ability to internalize albumin molecules, which results from the function of this protein as a basic source of energy and building blocks in rapidly dividing tumor cells [64]. Albumin retention in the tumor microenvironment is also facilitated by the increased expression of the glycoprotein SPARC (secreted protein, acidic and rich in cysteine), characteristic of solid tumors, an acidic secretory protein rich in cysteine, also known as osteonectin [65]. The first drug approved by the FDA in a form bound to albumin was Abraxane—a polymeric NP based on HSA (human serum albumin) and paclitaxel. Paclitaxel is a cytotoxic drug belonging to the taxane group, which inhibits mitotic divisions by binding to microtubules, thus disrupting the formation of the mitotic spindle. It is a drug with high efficacy, but its serious limitation is its strong hydrophobicity and the resulting need for emulsifiers to dissolve the drug in water. The most commonly used emulsifier was polyoxyethylated castor oil, which effectively increased the solubility of paclitaxel, but at the same time increased the risk of adverse effects associated with excessive histamine secretion as a result of hypersensitivity reactions, including anaphylaxis. The production of paclitaxel in combination with albumin allows avoiding the use of CrEL as a stabilizer, while increasing the safety of the therapy [66]. Clinical studies have shown that albumin-bound paclitaxel demonstrated higher response rates and better tolerability than solvent-based formulations in patients with metastatic breast cancer and advanced non-small cell lung cancer. Additionally, Abraxane has been shown to achieve 33% higher tumor uptake than traditional paclitaxel, which is explained by increased drug accumulation due to albumin uptake by SPARC. Abraxane is currently approved by the FDA for monotherapy for the treatment of metastatic breast cancer in adult patients who have failed first-line therapy for metastatic disease and who are not candidates for standard anthracycline therapy in combination with gemcitabine. It is also indicated for the first-line treatment of metastatic pancreatic adenocarcinoma in adults and in combination with carboplatin for the first-line treatment of non-small cell lung cancer in adult patients who are not candidates for radical surgery and/or radiotherapy [67].

Polymer NPs

Polymer NPs include two types of NCs—nanocapsules and nanospheres, which differ in terms of morphology. Nanocapsules are referred to as reservoir systems and consist of an oily core in which the drug is dissolved and a polymer shell responsible for controlling the release profile of the drug from the core. Nanospheres, considered a matrix system, have a structure of a continuous polymer network. The drug can be retained inside the polymer network or absorbed on its surface [68]. The polymers that form NPs can be linear, branched, or spherical, and their size is strictly controlled. The main synthetic polymers include polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polyglutamic acid, and N-(2-hydroxypropyl)-methacrylamide (HPMA) [69]. PLGA is one of the most commonly used polymers for the construction of NPs, partly due to its minimal systemic toxicity. This compound is hydrolyzed in the body to monomers in the form of lactic acid and glycolic acid, which are then metabolized in the Krebs cycle and removed as carbon dioxide and water [70]. PLGA NPs are commonly used to encapsulate various types of anticancer drugs, which allows for their more effective delivery in vivo. The most common forms of PLGA-based NPs are microspheres or microparticles [71]. They are used in oncology to transport many drugs, including VCR, cisplatin, etoposide, paclitaxel, DOX, curcumin, triptorelin, dexamethasone, rapamycin, and many others [72–79].

The main barrier to oncological treatment is the development of multidrug resistance (MDR). It appears after a longer period of chemotherapy and causes a decrease in the effectiveness of the treatment, which may consequently lead to the failure of the therapy. One of the most common mechanisms of resistance development is the P-glycoprotein-based pumping of the drug from the cancer cell. P-glycoprotein, encoded by the MDR-1 gene, exports a wide range of chemotherapeutic drugs, reducing their accumulation in tumor cells and thus leading to low treatment efficacy. For example, VCR sulfate is a widely used anticancer agent, but many cancer cells are resistant to its action due to the drug efflux mechanism via the P-glycoprotein complex-related pump. P-glycoprotein probably identifies only those drugs that are bound to the plasma membrane. VCR-loaded PLGA NPs, after internalization, usually end up in endolysosomal complexes, which allows them to escape from the drug efflux mechanism [80]. Additionally, the use of P-glycoprotein inhibitors such as verapamil (VRP), cyclosporine A, or quinidine could help overcome the resistance of cancer cells to treatment. In vivo studies compared the efficacy of PLGA NPs containing VCR and VRP (VCR-VRP-PLGANPs) with free VCR, free VCR/VRP combinations, and single-drug NP combinations against MDR breast cancer cells. A statistically significant difference in the rate of tumor growth inhibition was observed between the NPs and the other study groups. The slowest tumor growth was observed with VCR-VRP-PLGANPs. As expected, VRP increased the antitumor potency of VCR, and encapsulation of both substances into PLGA NPs further enhanced the effect of this drug combination, probably through increased accumulation of NPs in the tumor microenvironment and rapid release in the weakly acidic environment inside the tumor cells [81].

Dendrimers

Dendrimers are spherical molecules characterized by a symmetrical, highly branched three-dimensional structure. Their central part is a core, composed of a single atom or group of atoms, from which symmetrical branches called dendrons extend. At the ends of dendrimers, covalent or non-covalent attachment of biologically active molecules, drugs, genes, or contrast agents is possible. The structure of dendrons is largely responsible for the physical and chemical properties of dendrimers [82]. There are complete dendrimers, in the case of molecules with amine (–NH₂) or hydroxyl (–OH) end groups, and half-dendrimers, terminated with carboxyl groups (–COOH). The increase in the number of repeating branching units determines the generation of the dendrimer and is responsible for the formation of a circular structure. Therefore, the higher the generation of the dendrimer, the higher the density of the molecule packing, which is why it is possible to synthesize dendrimers only up to 10 generations. The high degree of control over the possible structure of the dendrimer makes these molecules attractive for delivering drugs to tissues. Therapeutic compounds can be enclosed in their cavities, bound to the surface via hydrophobic or electrostatic interactions, or covalently linked to end groups. There are different types of dendrimers, including phosphate dendrimers (P-dendrimers), polyamines, carbosilicates (CBS), polypeptides, polyesters, and the best known polyamidoamines (PAMAMs) [83].

Dendrimers derived from PAMAM are the most commonly used group of this type of NPs. They are hydrophilic, biocompatible, and non-immunogenic systems, which favors their use as drug carriers or nucleic acids [84]. In an in vivo study, the effect of conjugation of DOX with carboxyl end groups of PAMAM dendrimers on the efficacy of intrapulmonary therapy in a mouse model of melanoma lung metastases was assessed. The results showed a significant increase in the therapeutic efficacy of DOX, as indicated by a smaller number of nodules observed in the lungs and increased survival rates compared to the free form of DOX. Additionally, the intrapulmonary route of administration and conjugation with PAMAM allowed for a longer drug accumulation in the lungs compared to intravenous administration, while significantly reducing the distribution of DOX to the heart tissue [85].

CNTs

CNTs are cylindrical structures with a wide variety of structures. CNTs include two classes of nanomaterials, including single-walled CNTs (SWCNTs), composed of a single graphene layer rolled into a cylindrical tube, and multi-walled CNTs (MWCNTs), containing many concentric graphene cylinders. The diameter of MWCNTs, due to their multilayer structure, is usually larger than SWCNTs (approx. 1 nm in diameter) and ranges from 5 to 20 nm. CNTs, especially MWCNTs, have attracted attention due to their promising properties, such as large absorption surface area, high material strength, high charging capacity, optical and electrical properties, as well as high stability, biocompatibility, and the ability to release therapeutic agents [86]. In addition, they are considered to be “intelligent” materials, closely related to the mechanism of microtubule action. MWCNTs can penetrate many biological barriers, including the interior of cells, where they exhibit unique biomimetic properties with cytoskeletal elements, and primarily with microtubules, thus demonstrating a common anticancer effect with drugs, consisting of inhibiting the cell cycle in the mitotic phase. In vivo studies have shown the benefits of using MWCNTs as a drug carrier, due to the additional inhibition of the cancer cell cycle in the M phase. For example, a combination of 5-FU with a carrier in the form of MWCNTs (5-FU-MWCNT) was tested. 5-FU is a widely used anticancer drug, the mechanism of action of which is based on inhibiting the cell cycle in the S phase. A study conducted on a mouse model of melanoma showed the highest efficacy of 5-FU-MWCNT in reducing tumor mass compared to 5-FU monotherapy and MWCNT alone. These results confirmed the possibility of increasing the effectiveness of the treatment by using two complementary cytotoxic mechanisms generated by both therapies, disrupting two different stages of the cell cycle [87]. Carbon tubes are not ideal materials, however, due to their structure, they also have certain disadvantages, such as lack of biodegradability, tendency to accumulate and resulting toxicity. The toxic effect is mainly due to the similarity in structure to asbestos fibers. The side effects reported in studies include long-term inflammation, but also the development of neoplastic changes, mainly in the form of pleural mesothelioma (when inhaling MWCNT) [88]. Despite the great benefits of using MWCNT, toxic effects on specific cell types, such as alveolar epithelial cells or normal bronchial epithelial cells, have also been demonstrated [89]. However, the results of studies on the potential toxicity of MWCNT are inconsistent, hence, further work is required in this direction.

NPs in imaging diagnostics of cancer diseases

NPs can be used not only as drug carriers, but also as contrast agents, enabling imaging at the molecular level. More precise visualization of pathologically changed tissues is possible due to the small size of NCs, optical properties, and the ability to accumulate in the tumor area. Additionally, the use of specific ligands allows detection of pathological changes at the cellular level, allowing for earlier detection of changes, which in turn increases the probability of complete recovery of the patient [90].

The concept of theragnosis is becoming increasingly common in medicine. Theragnosis (from the words “therapy” and “diagnosis”) is a modern concept that combines diagnostics and therapy in one process. Multifunctional NPs can be combined simultaneously with diagnostic markers and therapeutic agents, thus allowing for detection, assessment of advancement, and simultaneous treatment. One of the most attractive materials in the context of theragnosis is AuNPs, which results from their properties of absorbing and scattering light [the phenomenon of surface plasmon resonance (SPR)]; additionally, they are characterized by biocompatibility and limited toxicity [91]. AuNP-based theragnosis has some limitations, but it is a promising perspective for the use of NPs for better diagnostics and treatment of cancer.

NDCDS

After demonstrating the effectiveness of anticancer drugs, various schemes of combined chemotherapy began to be constructed to increase therapeutic efficacy. In recent years, the focus has been on the mechanism of co-delivery of these drugs, combining molecules with different physicochemical and pharmacological properties using NDCDS nanotechnology. Thanks to this, it is possible to minimize the side effects of therapy and the toxicity of individual molecules. This delays tumor adaptation, and thanks to this, also limits the number of mutations occurring within it. This is a therapeutic option based on combination therapy, from which it differs, among other things, in the pharmaceutical compatibility of the drugs used—they can be mixed in one infusion preparation and administered together—conventional chemotherapy rarely provides such a possibility. Thanks to this, sequential and precise action of anticancer drugs with different target points is possible. The most commonly combined are DOX and paclitaxel molecules, which are administered in free form in a molar ratio of 5:1, while in the NDCDS formula the highest effectiveness was demonstrated by their combination in a ratio of 2:1. Zhang et al. [92] in their studies proved that the use of nanotechnology improves the effectiveness of inhibiting the growth of cancer cells compared to the use of separate supply of the same drugs in the same concentrations, but separately. High effectiveness of the above-mentioned has been demonstrated especially in the treatment of non-small cell lung cancer, and in the combination of cisplatin with DOX, paclitaxel, or rifampicin in the therapy of ovarian cancer and melanoma. An example of NDCDS is also the previously described polymer-lipid molecules combining drugs with both hydrophilic and hydrophobic character. NDCDS systems are subject to continuous research combining molecules of different sizes, including monoclonal antibodies, for example, co-delivery of trastuzumab with DOX, cisplatin, or paclitaxel in the case of breast cancer with overexpression of HER2/neu. A challenge in NDCDS research, apart from pharmaceutical compatibility, is the type of biomaterial from which the carrier for individual drugs is made. Mainly liposome and polymer materials are used, but also inorganic materials based on AuNPs [92]. DNA damage induced by cis-platinum (Cis-Pt) mediated chemotherapy was significantly impaired by the highly expressed programmed death ligand-1 in tumor cells [93]. The immune checkpoint blockade therapies that target the programmed cell death ligand-1 have been used as revolutionary cancer treatments in the clinic [94]. NPs with efficient tumor-targeting capacity, tumor-responsive prosperity, and versatility for combination therapy were identified as new avenues for programmed death ligand-1 targeting cancer immunotherapies [95].