Lipid-polymer hybrid nanoparticles in cancer therapy: current overview and future directions

Cancer remains one of the leading cause of death worldwide. Current therapies are still ineffective in completely eradicating the disease. In the last two decades, the use of nanodelivery systems has emerged as an effective way to potentiate the therapeutic properties of anti-cancer drugs by improving their solubility and stability, prolong drug half-lives in plasma, minimize drug’s toxicity by reducing its off-target distribution, and promote drugs’ accumulation at the desired target site. Liposomes and polymer nanoparticles are the most studied and have demonstrated to be the most effective delivery systems for anti-cancer drugs. However, both liposomes and polymeric nanoparticles suffer from limitations, including high instability, rapid drug release, limited drug loading capacity, low biocompatibility and lack of suitability for large-scale production. To overcome these limitations, lipid-polymer hybrid nanoparticles (LPHNPs) have been developed to merge the advantages of both lipid- and polymer-based nanocarriers, such as high biocompatibility and stability, improved drug loading and controlled release, as well as increased drug half-lives and therapeutic efficacy. This review provides an overview on the synthesis, properties and application of LPHNPs for cancer therapy.


LPHNPs: structural classification, properties, synthesis methods
In the design of nanocarriers for the delivery of anticancer agents, the most commonly used matrices are polymers and lipids, each of them with specific advantages associated with their use [1]. Among the polymeric nanosystems, we distinguish mainly polymeric NPs, polymeric micelles and polymer-drug conjugates, while among the lipid-based nanosystems we distinguish liposomes, solid lipid NPs and nanostructured lipid vectors [2]. Lipid-based nanocarriers offer several advantages such as a low production cost, greater trapping efficiency of the therapeutic agent, however they tend to display a reduced stability, a fast load release and high polydispersity [3]. In addition, there is a limited possibility for the chemical modification/functionalization of these nanostructures, thus reducing their application for active targeting approaches [4]. Polymeric nanosystems, on the other hand, are characterized by a high possibility of chemical modifications, an essential feature in the development of personalized therapies [5]. In addition, polymeric NPs offer other advantages such as the possibility of obtaining NPs of small dimensions and with reduced polydispersity, as well as diversity in the synthesis procedures, simple and reproducible synthesis process, and better stability [6]. However, polymeric nanosystems also display limitations, such as the use of organic solvents in the synthesis process, the toxicity linked to the degradation products of the polymer and the limited trapping capacity of drugs [7]. A new generation of nanosystems has been created to exploit the advantages of both polymeric systems and lipid-based systems, thanks to the production of a hybrid system composed of a polymeric core coated by a lipid shell, named lipid-polymer hybrid nanoparticles (LPHNPs) [8]. Such systems combine the advantages of lipid-based nanocarriers, such as increased drug carrying capacity, biomimetic nature and biocompatibility, with the advantages of polymeric nanocarriers such as desirable drug release profile and variety of surface chemical functionalization/modification. In the simplest design, LPHNPs consist of a hydrophilic or hydrophobic polymer core encapsulating the therapeutic agent of interest, surrounded by a lipid layer, which improves nanoparticles' biocompatibility and stability upon systemic administration [9][10][11]. Several polymers have been proposed for the development of LPHNPs, among the most widely employed are polycaprolactone (PCL), poly (lactic-co-glycolic acid) (PLGA), Polylactic acid (PLA), poly β-amino ester (PbAE), Chitosan [12,13], which are highly biocompatible and biodegradable polymers, in some cases approved by the Food and Drug Administration (FDA). Regarding the lipid shell, its composition can be tailored based on the type of therapeutic agent that is intended to be release, and the desired surface properties needed to achieve efficient uptake, controlled drug release, and biodistribution [14]. The outer layer properties can be easily modulated by changing the lipid composition and exploited to facilitate non-covalent or covalent attachment of antibodies, ligands, aptamers, and bioactive molecules such as DNA, RNA or proteins [15]. Additionally, charged or zwitterionic lipids are often preferred to promote electrostatic interactions between the lipid layers and the opposite charge of the polymeric core, thus promoting self-assembling of the nanostructure. Similarly, the preparation of a lipophilic core can favor its interaction with the hydrophobic tail domain of the lipids constituting the lipid layer [16]. Finally, pegylated lipids (PEG-lipids) can be included into the outer shell, to improve the circulation time of nanoparticles in the bloodstream since it can prevent the interaction with serum proteins [17].

Structural classification of LPHNPs
Based on their structure, LPHNPs can be classified as follows: (1) Monolithic hybrid nanosystems, (2) core-shell nanosystems, (3) Hallow core-shell nanoparticles, (4) Biomimetic lipid-polymer hybrid nanosystmes and (5) Polymer-caged liposomes [11]. The monolithic hybrid nanosystems ( figure 1(A)) are characterized by a polymeric matrix in which the lipid molecules are dispersed randomily [18]. core-shell nanoparticles ( figure 1(B)) are formed by a polymeric core that acts as a support for the deposition of a highly biocompatible lipid shell. A variant of this kind of nanovectors is represented by the hollow core-shell nanosystems (figure 1(C)), which possess a lipid layer composed of PEG-lipids and neutral lipids surrounding an inner polymer layer, which may interact with a cationic lipid layer, delimiting a hallow inner aqueous core [19]. The biomimetic lipid-polymer hybrid (figure 1(D)) nanosystems are composed of a polymeric core coated with cellderived membranes [20]. The last class of LPHNPs are the polymer-caged liposomes constituted by an external polymer layer anchored on the surface of liposomes (figure 1(E)) [21].

Properties of LPHNPs
LPHNPs offer the possibility to integrate in the same nanocarrier the advantages of both polymeric and lipid delivery vectors, leading to the realization of a new generation of nanoformulation with improved physical, chemical and functional properties [22]. Due to their composition, LPHNPs display good biocompatability, and the entire nanovector can be potentially metabolized by the body. They also possess a large surface area available for conjugation with drugs or ligands, and their morphology along with surface properties can be tuned by varying the composition of the core and lipid shell [23], enabling a greater entrapment efficiency and a controlled release of the therapeutic agents. Furthermore, LPHNPs tend to display a high mechanical stability both during storage and in serum, thanks to the polymeric core which acts as a cytoskeleton and to the PEGcoated lipid layer, which improves their circulation time in vivo by eluding the immune system [24][25][26][27][28]. LPHNPs of core-shell type with dimensions equal to or less than 100 nm allow the intracellular release of the drug causing reduced cytotoxicity [29]. Finally, thanks to the amphiphilic character of the lipids, hydrophilic molecules can be adsorbed on the lipid bilayer, while hydrophobic molecules can be trapped at the level of the hydrophobic lamellar region. Thus, LPHNPs can trap and co-administer multiple hydrophilic and hydrophobic therapeutic agents, acting as a universal cancer therapy platform [30,31]. Figure 2 summarizes the main advantages of LPHNPs.

Synthesis methods for LPHNPs
Various methods have been developed for the synthesis of LPHNPs, but they can be broadly grouped in two main strategies: (1) Two-step methods and (2) One-step methods [32], see figure 3.

Two-step methods
The approaches proposed for the synthesis of LPHNPs in the initial studies, were based on a two-step method, wherein polymeric nanoparticles are mixed with the prepared liposomes, and the incorporation of the lipid shell onto the surface of the polymer core is promoted by electrostatic forces [33]. Otherwise, the prepared polymeric nanoparticles can be added to dried lipid film. In either case, the assembling of the two components is promoted through the introduction of energy via vortexing and/or ultrasonication and heating at a temperature above the phase transition temperature of the lipid layer [34]. The polymeric core can be prepared by nanoprecipitation, emulsification-solvent evaporation or high-pressure homogenization [35]. A purification step can be introduced to ensure the removal of lipidic or polymeric nanoparticles and obtain the isolation of LPHNPs [36].
1.3.2. One-step methods Two-step methods have shown to be time-consuming and poorly suitable for their scaling-up [37]. Therefore, large efforts have been dedicated to the development of more efficient synthesis methods that can allow obtaining LPHNPs in a single step [38]. Usually, most of the previously reported one-step methods are based on  the nanoprecipitation technique or emulsification-solvent evaporation techniques [39]. However, nanoprecipitation has shown important advantages over emulsification-solvent evaporation procedure, such as simplicity, and the possibility of obtaining nanoparticles with a narrow size distribution [40]. Furthermore, nanoprecipitation only requires the use of non-toxic solvents (e.g. ethanol), and it involves a procedure that can be automated using a microfluidic platform and, in some cases, allows a superior loading efficiency, especially with macromolecules (i.e. mRNA, pDNA and protein) [41]. In the conventional nanoprecipitation technique, an organic phase, containing the lipophilic drug and polymer dissolved in a water-miscible organic solvent (Acetone, ethanol, methanol, etc), is mixed with the lipid/PEG-lipid dissolved in water. The aqueous phase is heated above its gel-to-liquid transition temperature in a pre-mixing step, in order to prepare a homogeneously dispersed lipid solution [42]. The mixing of the organic phase with the aqueous phase triggers the reorganization of the polymer into nanoparticles and the simultaneous self-assemblage of the lipids surrounding the polymer core, where the hydrophobic tails of the lipids are directed toward the inner side and the hydrophilic head groups face out toward the external aqueous solution [43]. The removal of the organic phase can be ensure by evaporation, dialysis or ultrafiltration. Other one-step approaches have been previously reported. For instance, Fang et al showed ultrafast synthesis (∼5 min) of small and well monodisperse LPHNPs. About a decade ago, Valencia et al demonstrated that LPHNPs with relatively narrow size distribution can be prepared in microfluidic reactors, where the organic and aqueous phases are rapidly mixed together [44]. In the second decade of the 21st century, Kim et al reported a pattern-tunable microvortex platform for large production (∼3 g hour −1 ) of LPHNPs with a controllable size (∼30-170 nm) and low PDI (∼0.1) [45].

Application of LPHNPs in cancer therapy
In the last two decades, the use of nanodelivery systems has emerged as an effective way to potentiate the therapeutic properties of anti-cancer drugs by improving their solubility and stability, minimize drugs' toxicity by reducing its off-target accumulation [46,47]. Many efforts are currently concentrated on the design of formulations capable of a highly specific interaction with cancer cells to improve the therapeutic efficacy of conventional cancer therapies [48]. Recently, several research groups have focused on the development of nanoplatforms capable of delivering the different anticancer therapies in a targeted way while sparing healthy cells. These nanostructures must be able to mantain their integrity while on circulation and release their cargo in a controlled manner once the desired site is reached [49]. LPHNPs compared to other nanosystems, such as lipid-based systems and polymeric NPs, have numerous advantages, resulting in valid alternatives in the treatment of cancer [50]. The main advantage of the use of LPHNPs in the administration of anticancer agents is represented by the possibility of incorporating therapeutic agents with different physicochemical properties, thanks to their hybrid structure characterized by a polymer and lipid components that can possess distinct properties [51]. In addition, hydrophobic drugs can be co-administered with emerging therapeutic agents such as nucleic acids, proteins and peptides. In this regard, a possible approach is to encapsulate the hydrophobic drugs into the lipophylic polymeric core, while charged biomolecules are conjugated or adsorbed to the lipid shell [52,53]. Several studies have confirmed a greater drug encapsulation efficiency of LPHNPs compared to polymer nanomatrixes, as well as greater stability with non-significant drug losses and controlled load release kinetics [54]. In this regard, the group of Wong et al reported that LPHNPs characterized by SLN (solid lipid NPs) that coat a negatively charged polymer for the administration of salidroside (Sal), a positively charged hydrophilic drug [55]. Sal is a water-soluble anticancer drug, and in this study the authors attain its encapsulation exploiting its affinity with the hydrophobic portion of the outer lipid membrane of the hybrid vector. With this strategy, the authors generated small nanoparticles with high loading capacity, able to efficiently target the tumor site upon administration [55]. There are several limitations related to the use of anticancer drugs, such as unwanted biodistribution, rapid drug clearance and non-targeted action, leading to a series of adverse effects on non-diseased tissues [56,57]. A controlled and selective administration of therapeutic agents will lead to a significant reduction of adverse systemic effects. Due to their structure, LPHNPs are particularly suitable for the conjugation of ligands directed towards the over-expressed receptors on tumor cells, thus improving the tumor targeting and maximizing the therapeutic action of the administered agents towards tumor cells [58,59]. Different types of ligands, such as small molecules, peptides, antibodies and aptamers have been employed for the functionalization of the outer surface of hybrid nanoparticles in active targeting strategies [60]. Such targeting ligands are directly bound on the outermost surface of LPHNPs through electrostatic interactions or through strong covalent attachment [61]. The internalization by tumor cells of hybrid nanoformulations decorated with targeting ligands occurs through a mechanism of receptor-mediated endocytosis, with significantly higher affinity for the target tissue and lower affinity for non-diseased tissues [62]. For example, hybrid nanosystems with a core-shell structure have been produced using a natural polymeric composite of chondroitin sulfate and chitosan coated with a PEGylated lipid layer in order to increase stability [63]. In addition, the lipid layer was functionalized with Folic acid (FA) for targeted delivery to cancer cells. The results obtained from the uptake studies performed using fluorescently labeled nanopartilces revealed that functionalization with FA enhanced nanoparticles' internalization by cancer cells. In addition, active targeting also resulted in a prolonged release of the therapeutic agent (sorafenib) with a consequent enhancement of the anti-tumor effect [64]. Similarly, another study reported a folate-decorated LPHNP, synthesized using the lipid DSPE-PEG2000 and a PCL-PEG-PCL poly (ε-caprolactone) -poli (ethylene glycol) -poli (ε-caprolactone) copolymer) to constitute the core of the NPs, for the prolonged, controlled and targeted release of paclitaxel (PTX) [65]. In this study, the folate receptor positive mouse carcinoma cells (EMT6 cells) displayed a greater uptake of the folate-functionalized LPHNPs compared to the folate receptor negative cells (L929 fibroblasts); thus confirming that the internalization of nanoformulations in EMT6 tumor cells is achieved through receptormediated endocytosis. Furthermore, in vitro cytotoxicity tests confirmed that folate-functionalized and PTXloaded LPHNPs have a two times greater cytotoxic effect over non-targeted LHPNPs. This higher cytotoxic effect was also confirmed in vivo, as tumor-bearing mice treated with folate-functionalized LHPNPs showed a superior tumor growth inhibition compared to mice treated with non-functionalized LHPNPs [65,66]. In another study conducted by the group of Gu et al a hybrid nanofurmulation was developed to overcome the drawbacks associated to the use of cisplatin and Indocyanine green (ICG), a photosensitizer used for clinical imaging and phototheramal. The authors proposed a single-step sonication method for the manufacturing of folate-modified, cisplatin/ICG-loaded LPHNPs. The LPHNPs exhibited optimal monodispersity and stability, as well as excellent near infrared (NIR) penetration ability. The folate-functionalized LPHNPs showed an improved targeting efficacy in MCF-7 tumor cells over-expressing folate receptors (FR), compared to FRnegative A549 tumor cells. In addition, the functionalized formulation was more effective than free PTX or ICG treatment alone at mediating apoptosis and necrosis of MCF-7 cells, thus proving LPHNPs may be a suitable kind vector for multimodal tumor-targeted therapy [67,68].
In line with the above, in a recent study Yugui et al applied core-shell LPHNPs functionalized with FA and loaded with an gefitinib, an EGFR inhibitor, and a radioisotope (yttrium 90) for the development of an effective combination treatment for nasopharyngeal cancer [69]. These FR targeted nanosystems demonstrated a synergistic chemoradiotherapy effect, with improved antitumor efficacy in vivo and 90% drug accumulation at the desired site [69]. In a similar study Guo et al through the use of hybrid nanoparticles consisting of a PLGA polymeric core and a lipid shell functionalized with transferrin (Tf), demonstrated the antiproliferative action of DOX. Tf ligands were conjugated to the lipid shell via a post-insertion process. The hybrid nanoformulations were internalized by A549 cells through TfR-mediated endocytosis, with an efficiency 2.8-4.1 times greater than PLGA polymeric nanoparticles. In addition, the release rate of the hybrid nanoparticles was lower than that of the non-hybrid ones, pointing out that hybrid nanovectors can promote a prolonged and sustained drug release [70]. A similar approach was applied in the design of a hybrid nanosystem formed by PLGA coated with a lipid shell, produced through the solvent injection technique, for the delivery of an aromatase inhibitor. [71]. This hybrid nanosystem demonstrated a 36.6-fold greater trapping efficiency of the aromatase inhibitor (7α-(4′ amino) phenylthiol-1,4-androstadiene-3,17-dione (7α-APTADD) and an improved therapeutic action thanks to the functionalization with the Tf ligand, thus achieving a targeted delivery. Indeed, the nanovector showed a high uptake in SKBR-3 cells human breast cancer cells through TfR-mediated endocytosis [71]. The prolonged release of 7α-APTADD demonstrated a dose-dependent inhibition of aromatase with superior accumulation in tumor mass, which confirmed effective inhibition of targeted aromatase inhibitors versus that of a non-targeted hybrid system [71]. Current advances in cancer research have revealed that biomolecules such as nucleic acids (DNA and RNA), proteins and peptides have shown an important therapeutic potential in the treatment of cancer, when administered together with other drugs for a synergistic therapeutic effect, constituting a promising strategy against chemoresistance [72]. However, the co-encapsulation and release of hydrophobic drugs, such as conventional chemotherapy drugs, together with these biomolecules presents a major challenge, which may be overcome with hybrid nanoparticles [73]. Sengupta et al, in this regard, developed a hybrid nanoparticle with a diameter of 180 and 200 nm, consisting of a central block polymer core coated with a pegylated phospholipid [74]. DOX, cytotoxic drug, was conjugated to the polymer core, and an anti-angiogenic agent, combretastatin-A4, was then, entrapped in the lipid layer. The lipid shell was disrupted within the tumor leading to the rapid release of combretastatin-A4 and a consequent vascular collapse [74]. All this led to the retention of intra-tumoral retention of the nanoparticles with subsequent release of DOX from the PLGA-DOX oligomer for the killing of tumor cells [74]. This study highlighted the versatility offered by hybrid core-shell nanosystems in the application of combinatorial therapies. The group of Aryal et al followed a similar strategy by encapsulated Paclitaxel-cisplatin conjugate, chemically conjugated through the glutaric acid linker, into hybrid LPHNPs. Non-encapsulated drug conjugate versus drug conjugates trapped in LPHNPs demonstrated reduced cytotoxicity towards a human ovarian cancer cell line [75]. The nanoparticles facilitated the internalization of drug conjugates through endocytosis, overcoming the limitation of their transport ability across the lipid bilayer. LPHNPs have been employed for the co-administration of chemotherapeutic agents in combination with radioisotopes, nucleotide sequences, proteins and diagnostic agents [76]. In a recent study, the selective coadministration of DOX with pDNA encoding GFP was realized through a hybrid nanosystem consisting of a hydrophobic polymer core of PLGA and a self-assembled PEGylated lipid coating coated with hydrophilic folate. In this system, DOX was trapped in the polymer core while the cationic lipid shell was employed to bind the DNA via electrostatic interactions [77]. LPHNPs have been recently applied for cancer immunotherapy, and they may be particularly suitable for the development of combinatorial immunotherapies targeting different immune escape mechanisms. A hybrid nanoformulation was proposed by Park et al, consisting of liposomal polymeric gels of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers for the delivery of a small hydrophobic molecular inhibitor of transforming growth factor β (TGF-β), and watersoluble protein cytokine, interleukin-2 (IL-2), to the tumour microenvironment for the treatment of metastatic melanoma [78]. The results obtained showed a significant delay in tumor growth with an increase in survival time of tumor-bearing mice, providing evidence of the efficacy of hybrid nanovectors for multi-drug delivery [78]. Overall, in the various studies reported the co-administration strategies resulted in significant benefits for an improved tumor inhibition potential compared to single drug administration, and a reduction in drug chemoresistance. Table 1 shows several lipid-polymer hybrid nanosystems used for the delivery of anticancer agents.

Future directions and conclusions
The relative high loading capacity for different therapeutic agents, excellent stability in the bloodstream and drug cargo-transporting capability in vivo, renders LPNNPs a highly appealing delivery vehicle for cancer therapeutics. In addition to numerous other advantages such as improved biocompatibility, long circulation time in the body, targeted targeting, adjustable drug release kinetics and reduced drug degradation thanks to the external PEGylated lipid layer, both compared to liposomes and polymer NPs, believed to date the nanostructures of choice for the administration of anticancer therapies. All these features reveal the extraordinary potential of LPHNPs as versatile delivery platforms with unique therapeutic potential in the treatment of cancer. The LPHNPs as a whole represent structures with adjustable characteristics in terms of release kinetics, which allow the administration of an optimal quantity of therapeutic agent in the desired site with limited adverse effects off target. Numerous ligands that target proteins over-expressed by tumor cells have been explored for the functionalization of hybrid NPs, frequently reporting an improved accumulation of the therapeutic agent in the tumor. Therefore we believe that future efforts should focus on programming in vivo experiments that lead to the optimization of the ligand density, with the aim of improving the pharmacokinetics of NPs. Furthermore, LPHNPs are capable of encapsulating a large variety of therapeutic agents due to their unique structure. These nanocarriers can trap multiple drugs (co-encapsulation) by exploiting their synergistic effect. Consequently, another interesting prospect would be the optimization of loading protocols in order to improve the co-entrapment efficiency of drugs with different properties (hydrophilic and hydrophobic), considering that the co-administration strategy is proving to be more and more a promising approach in the treatment of incurable diseases, such as cancer. Several studies have highlighted the unique therapeutic potential of LPHNPs loaded with two drugs. In this regard, the control of the molar ratio of the two drugs and their loading efficiency is fundamental. Many lipid-based medications have been and are currently tested in clinical trials, and a good number is already commercially available. However, the clinical implementation of polymeric NPs remains limited, as there are still concerns regarding their safety profile. Preclinical studies have provided evidences that the use of LPHNPs can offer the potential to integrate the advantages of both lipid-and polymeric-based systems in the same platform and overcome the major limitations of polymeric NPs related mostly with toxicity concerns. Indeed, the introduction of an outer layer lipid shell could consent to modulate properties like hydrophobicity, stimuli-triggered drug release, targeting efficiency and polymer chargeassociated toxicity. However, the implementation of large-scale production procedures and the selection of materials with a good biocompatibility represent the major obstacles for their introduction into the clinic. For instance, there is a need for a proper selection of solvent to be utilized during synthesis, as this can significantly affect the stability of the NPs and its inefficient removal may induce undesired toxicity issues.
Once challenges related with the scale-up of the manufacturing procedures are addressed, more exhaustive studies both in preclinical and clinical settings are needed to allow the full exploitation of LPHNPs in cancer treatment. Guo et al have recently showed that nanoparticles' rigidity is a tunable feature in LPHNPs, which can be opportunely modulated to enhance tumor delivery efficiency by improving their tumor accumulation and uptake by target cells. However, still little is known about the influence of this parameter in defining the interaction of LPHNPs with immune cells and how this aspect can be eventually harnessed to potentiate the effectiveness of cancer immunotherapies [103]. We conclude our review by stating that LPHNPs constitute an intelligent and attractive platform for anticancer therapy since multiple pharmacological action and targeted Showed decreased in vivo clearance of (S)-Rg3 and prolonged circulation in the blood stream administration of the therapeutic agent are required in cancer, hence the ability and versatility to be designed as nanosystems of co-delivery opens a new path to the development of personalized and non-invasive nanotherapies with the potential to significantly improve patients' quality of life. Further explorations in the near future will lead to more exciting developments in the application of LPHNPs as nano-platforms for the targeted and safe transport of anticancer therapies.