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Review

New Applications of Lipid and Polymer-Based Nanoparticles for Nucleic Acids Delivery

by
Adelina-Gabriela Niculescu
1,
Alexandra Cătălina Bîrcă
1 and
Alexandru Mihai Grumezescu
1,2,3,*
1
Department of Science and Engineering of Oxide Materials and Nanomaterials, University Politehnica of Bucharest, 011061 Bucharest, Romania
2
Research Institute of the University of Bucharest—ICUB, University of Bucharest, 050657 Bucharest, Romania
3
Academy of Romanian Scientists, Ilfov No. 3, 50044 Bucharest, Romania
*
Author to whom correspondence should be addressed.
Pharmaceutics 2021, 13(12), 2053; https://doi.org/10.3390/pharmaceutics13122053
Submission received: 29 October 2021 / Revised: 26 November 2021 / Accepted: 29 November 2021 / Published: 1 December 2021
(This article belongs to the Special Issue Novel Nanoparticles for Targeted Delivery of Therapeutic Nucleic Acids)
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Abstract

:
Nucleic acids represent a promising lead for engineering the immune system. However, naked DNA, mRNA, siRNA, and other nucleic acids are prone to enzymatic degradation and face challenges crossing the cell membrane. Therefore, increasing research has been recently focused on developing novel delivery systems that are able to overcome these drawbacks. Particular attention has been drawn to designing lipid and polymer-based nanoparticles that protect nucleic acids and ensure their targeted delivery, controlled release, and enhanced cellular uptake. In this respect, this review aims to present the recent advances in the field, highlighting the possibility of using these nanosystems for therapeutic and prophylactic purposes towards combatting a broad range of infectious, chronic, and genetic disorders.

1. Introduction

The recent investigation of a multitude of therapeutic nucleic acids (TNAs) has paved the way for developing new treatment strategies for various wounds, infectious diseases, and chronic conditions. TNAs, such as plasmid DNA (pDNA), small interfering (or silencing) RNA (siRNA), self-amplifying RNA (saRNA), microRNA mimics, anti-microRNA oligonucleotides, messenger RNA (mRNA), and antisense oligonucleotides (ASOs), can be employed for creating therapeutics and vaccines that provide a cell-mediated immune response [1,2,3,4,5,6,7].
TNAs have gained increasing scientific interest, especially due to the long-lasting effects they offer in contrast to conventional treatments. Specifically, traditional medication induces temporary effects as it targets proteins instead of the underlying causes. In comparison, TNAs have the potential to produce long-term and even curative effects by gene inhibition, addition, replacement, or editing [2].
However, the direct delivery of nucleic acids has several drawbacks as naked nucleic acids are prone to enzymatic degradation, renal clearance, and poor cellular uptake as they face difficulties in crossing the cell membrane [4,8,9,10]. These challenges are mainly due to TNAs’ large molecular weight, negatively charged backbone, and more fragile and immunogenic potential than their oligonucleotide counterparts [6,11]. Thus, the clinical translation of such treatments is highly dependent on the delivery technologies that must enhance TNAs’ stability, protect against extracellular degradation, facilitate cell internalization, and improve target affinity [2,3,8].
In this respect, a broad range of delivery systems has been studied. Delivery vehicles such as lipid nanoparticles, polymeric micelles, dendrimers, polymer-based nanoparticles, hydrogels, polyplexes, proteins, and inorganic nanomaterials are under investigation [1,12]. Out of this plethora of possibilities, lipid nanoparticles are the most clinically advanced [11,13,14]; however, polymer and polymer–lipid hybrid particles have also recently become important categories of carrier platforms [15,16,17,18]. Moreover, such delivery systems have become highly attractive due to their targeting potential as they can be surface functionalized to allow accumulation and payload release in specific tissues [11,19,20].
In this context, this paper aims to provide an overview of the newly developed lipid and polymer-based nanosystems for nucleic acid delivery, emphasizing their importance in combatting various infectious, chronic, and genetic disorders.

2. Lipid-Based Delivery Systems

Lipid nanoparticles (LNPs) have attracted considerable research interest for encapsulating TNAs, especially due to their ionizable lipid presence, which is cationic at a low pH, thus, allowing complexation with negatively charged RNA or DNA [21,22]. Various generations of lipid nanocarriers have been tackled for TNAs delivery, including liposomes, solid lipid nanoparticles, nanostructured lipid carriers, and cationic lipid–nucleic acid complexes [23]. A brief history of how lipid-based RNA delivery systems evolved until the date is represented in Figure 1.
LNPs have also been extensively studied due to the relatively easy and scalable manufacturing processes they can be obtained through [3]. Specialized manufacturing and control techniques ensure the complex morphology and tailored lipid components and proportions required to create functional and efficient LNP-based delivery systems [11]. Examples of conventional synthesis methods include high-pressure homogenization [25,26], solvent emulsification–evaporation [27], ethanol injection nanoprecipitation [28], freeze-drying [29], and preformed vesicle method [30] (Figure 2).
For instance, Gomez-Aguado et al. [27] used the solvent emulsification-evaporation method to develop different solid lipid nanoparticles (SLNs) that combine cationic and ionizable lipids for the delivery of mRNA and pDNA. Their comparative study showed a higher percentage of transfected cells in mRNA formulation than for particles containing pDNA, especially in human retinal pigment epithelial cells (ARPE-19). Nonetheless, pDNA delivery resulted in greater protein production per cell in the mentioned cell line. Among the tested lipid formulations, SLNs containing only 1,2-dioleoyl-3-trimethylammonium-propane are considered the most promising for TNAs delivery.
Other synthesis methods were used by Wang et al. [29], who have created aerosolizable dry powder of lipid nanoparticles by thin-film freeze-drying (TFFD), spray drying, and conventional shelf freeze-drying. The researchers comparatively evaluated the obtained powders and tested the feasibility of engineering SLNs using the TFFD method, concluding that this synthesis strategy leads to better aerosol performance properties than the powders obtained otherwise.
However, some of the newest approaches for synthesizing LNPs are based on the direct mix of the organic phase (containing the lipids) with the aqueous phase (containing the TNA) using a microfluidic device [32]. Microfluidics represents a robust, scalable, and reproducible synthesis method [33]. This strategy has several advantages compared to conventional techniques as it allows the strict and easy control of flow rates, rapid mixing of the phases, and decreased synthesis time, resulting in nanoparticles with a small size, high monodispersity, and high encapsulation efficiency [28,34].
For instance, Bailet-Hytholt et al. [21] have synthesized LNPs encapsulated with mRNA or pDNA by means of a precise microfluidic mixing platform. Their experimental setup allowed the self-assembly of nanoparticles under laminar flow, benefiting from advantages such as reproducibility, speed, and low volume screening. Microfluidic methods were also employed by Roces et al. [33], who have prepared PolyA, ssDNA, and mRNA encapsulated LNPs in a Y-shape staggered herringbone micromixer (Figure 3). In this manner, the researchers obtained a consistent range of particle sizes, with dimensions below 100 nm, narrow size distribution, spherical shape, good stability, and high encapsulation efficiencies.
In what concerns their applicability, numerous lipid-based TNA delivery platforms have been recently constructed as potent novel platforms for cancer immunotherapies [35]. In particular, the use of mRNA formulations for different types of solid tumors and hematological malignancies is currently examined by various studies and even clinical trials [28]. Targeted LNPs can deliver RNA-based therapeutics to leukocytes to allow the precise and modular manipulation of gene expression, thus being a highly promising approach for many immune-related disorders, such as cancer, autoimmunity, and susceptibility to infectious diseases [36].
TNAs can also be employed in formulations for the treatment and prevention of cardiovascular diseases [37,38]. Clinical trials have investigated ASOs for lowering low-density lipoprotein cholesterol (LDL-C) [39], lowering transthyretin (TTR) blood levels [40,41,42], reducing atrial fibrillation burden [43], reducing serum lipoprotein (a) levels [44], and preventing neovascular glaucoma [45]. mRNA has also been evaluated for cardiovascular conditions, with several LNP-based formulations being tested for enhancing cardiac function and regeneration [46], reducing LDL-C levels [47], and treating endotheliopathy associated with vascular senescence [48,49]. Similarly, siRNA lipid-based formulations have been clinically tested for TTR-mediated amyloidosis [50] and hypercholesterolemia [51].
Another highly researched application of LNPs is for the development of COVID-19 vaccines, out of which several formulations have already entered clinical use for mRNA delivery [16,52,53,54,55]. Generally, these vaccines work on the principle of encoding the spike glycoprotein of SARS-CoV-2 (Figure 4) and directing the immune system against these antigens [56,57]. Clinical trials demonstrated the efficacy of LNP-mRNA formulations and an acceptable safety profile, leading to their approval for mass immunization [58,59].
COVID-19 vaccines are the most known current examples that make use of LNPs encapsulated with TNA, but this approach can be used for other infectious diseases as well [37,61,62,63,64,65,66,67]. TNA-LNP formulations have been evaluated against Zika virus [68], influenza [69,70], cytomegalovirus [71,72], chikungunya virus [73], hepatitis B [74,75,76], hepatitis C [77,78,79], human immunodeficiency virus (HIV) [80,81], and genital herpes [82].
The development of nonviral platforms for prenatal TNA delivery has recently emerged among the scientific research hot topics as a strategy to treat diseases before the onset of irreversible pathology and a non-invasive alternative to difficult-to-perform surgeries. What is more, the small size of a fetus compared to a postnatal recipient is a key factor for maximizing the delivery vector titer per weight of the recipient, thus, facilitating gene transduction [37,83]. In this regard, Riley et al. [84] have recently developed a library of ionizable LNPs for in utero mRNA delivery to mouse fetuses. Their LNPs successfully accumulated within fetal livers, lungs, and intestines, with higher therapeutic efficacy and safety than reference delivery systems (i.e., DLin-MC3-DMA and jetPEI), being promising candidates for protein replacement and gene editing platforms.
A series of examples of potential applications of lipid-based TNA delivery systems have been synthesized in Table 1 to summarize the above discussion and emphasize the versatility of such formulations.

3. Polymer-Based Delivery Systems

Cationic polymers have attracted interest for the development of innovative TNA carriers. The main reasoning is that they form electrostatic nanocomplexes with nucleic acids, which are highly negative, to facilitate their uptake by targeted cells. Alternatively, other hydrophobic polymers can physically entrap TNAs within nanoparticles [4].
The first polymer tested as a vehicle for in vitro-transcribed mRNA was diethylaminoethyl (DEAE) dextran. However, after it was proven that lipid-mediated mRNA transfection is 100 to 1000 times more efficient than DEAE-dextran, the evolution of polymeric carriers has been stalled, the attention shifting towards creating perpetually improved lipid-based nanosystems [111].
Nonetheless, after some time, research started being oriented towards developing natural and synthetic polymer-based nanosystems for TNAs delivery as an alternative for cancer treatments and non-viral vaccine formulations. Polymers such as hyaluronic acid [94,112,113,114], chitosan [94,113], DEAE dextran [111], poly-L-lysine (PLL) [94], poly-beta-amino-esters (PBAE) [111], polyethylenimine (PEI) [94,111], dimethylaminoethyl methacrylate (DEAMA) [111], poly(ethylene glycol) methacrylate (PEGMA) [111], and DEAEMA-co-n-butyl methacrylate [111] represent promising candidates for various TNAs delivery platforms.
Another interesting possibility is to combine two or more polymers in the same delivery system. For instance, Lü et al. [115] have developed antibody (Ab)-conjugated lactic-co-glycolic-PEI nanoparticles (LGA-PEI NPs) for the active-targeting delivery of TNAs. The research group obtained promising results for pancreatic cancer cells delivery both in vitro and in vivo, concluding that the synthesized carriers can be employed for other types of cancers as well if other specific biomarkers are targeted.
Polymers have also been tested for microneedle-based TNA delivery [111]. Koh et al. [116] have fabricated an mRNA-loaded dissolving microneedle patch from polyvinylpyrrolidone (PVP) for cutaneous TNA administration. This RNA-patch can mediate in vivo transgene expression for up to 72 h, the transfection efficiency and kinetics being comparable to subcutaneous injection. Moreover, this innovative delivery system induced higher cellular and humoral immune responses than subcutaneous injection. A similar administration strategy was used by Pan et al. [117], who have delivered signal transducer and activity transcription 3 (STAT3)-targeting PEI-encapsulated siRNA through dextran-hyaluronic acid dissolving microneedles. The researchers obtained promising results as the microneedles effectively penetrated and rapidly dissolved into the skin. In addition, the delivered complex successfully suppressed the development of melanoma by silencing the STAT3 gene.
To briefly summarize the current research progress, several examples of polymer-based TNA delivery systems and their applications have been gathered in Table 2.
Although various types of polymers and copolymers have been tested for TNAs delivery, the poorly understood correlation between their structure and their biological response hinders their development. Moreover, the polydispersity and difficulty in metabolizing large molecular weight polymers make them less appealing than LNPs. Their clinical application is also impeded by potential toxicity, colloidal instability, and relatively poor transfection efficiency [64,111]. Therefore, more research is required for understanding the influence of polymers’ chemical structure on their biological activity and optimally modulating their properties to overcome current challenges.

4. Lipid-Polymer Hybrid-Based Delivery Systems

Hybrid delivery systems have recently appeared as an alternative solution for overcoming the limitations of each individual lipid or polymer component. Specifically, the adverse effects of ionizable lipids (e.g., potential immune activation, cytotoxicity) are commonly mediated by shielding the LNPs with polyethylene glycol (PEG) [11].
For example, Sanghani et al. [123] have PEGylated LNPs made of pH-sensitive cationic lipid CL4H6 to safely and efficiently deliver myocardin-related transcription factor B (MRTF-B) siRNA into human conjunctival fibroblasts. A similar hybrid approach on TNA delivery was taken by Scmendel et al. [20], who have developed folate-containing lipoconjugates with PEG spacers incorporated into a liposome. These delivery systems exhibited higher transfection efficiency compared to non-targeted liposomal formulations and the commercial agent Lipofectamine 2000. Improved transfection efficiency was also obtained using the hybrid nanoparticles created by Siewert et al. [124]. The researchers have developed vehicles for mRNA delivery using different proportions of the cationic lipid DOTAP and the cationic biopolymer protamine, reporting significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. Another mRNA hybrid carrier was created by Xiong et al. [125]. This research group developed theranostic dendrimer-based LNPs containing PEGylated BODIPY dyes that combine mRNA delivery and NIR imaging, holding great promise for cancer’s simultaneous detection and treatment.
Zhou et al. [126] have synthesized a series of linear-dendritic PEG lipids (PEG-GnCm) to investigate the effect of modulating their hydrophobic domain for siRNA delivery as the surface component of dendrimer lipid-based nanoparticles (DLNPs). The researchers used different lipid alkyl lengths (C8, C12, C16) and different dendrimer generations (G1, G2, G3), creating vehicles with different physical properties and anchoring potential. These alterations did not affect particle size, RNA encapsulation, and stability but had a huge impact on delivery efficacy. PEG-G1C8, PEG-G1C12, PEG-G1C16, and PEG-G2C8 could effectively deliver siRNA in vitro and in vivo; however, all the other tested formulations lost their delivery ability, as the escape of DLNPs from endosomes at early cell incubation times was affected.
Furthermore, by tailoring the surface charge of LNPs, they can be endowed with targeting ability. According to the comparative study performed by Gabal et al. [127], anionic nanostructured lipid carriers exhibited 1.2 times higher targeting efficiency in the brain than their cationic counterparts. Thus, such anionic particles hold promise as carriers for brain disorders therapeutics. Nonetheless, anionic formulations have not faced the same development because of difficulties encountered in nucleic acid packaging and poor transfection efficiency. To overcome these challenges, Tagalakis et al. [128] have used cationic targeting peptides as a bridge between the PEGylated anionic liposomes and the pDNA freight. The newly developed structures demonstrated improved tissue penetration, enhanced dispersal, and more widespread cellular transfection than cationic systems. Similar anionic targeting hybrid nanocarriers have also shown promising results for siRNA delivery to neuroblastoma tumors with reduced systemic and cellular toxicity [129].
Another polymer that can be used in combination with LNPs is poly(lactic-co-glycolic acid) (PLGA). In this respect, Yang et al. [130] have developed a PLGA-LNP hybrid delivery system loaded with CRISPR/Cas9 plasmids targeting the MGMT gene and modified with the cRGD peptide. This system was constructed to open the blood–brain barrier (BBB) and ensure targeted gene delivery in vivo under focused ultrasound (FUS) irradiation. The obtained results emphasized a synergic targeting ability of the physically site-specific characteristics of FUS and the biologically active targeting ability of cRGD peptide, recommending this innovative carrier as a candidate for central nervous system TNA delivery.
A recently approached strategy for designing performant hybrid carriers is the creation of lipopolyplexes (LPPs), complexes combining nucleic acids with lipids and polymers (Figure 5). Such structures are promising delivery systems for gene therapy, especially due to their compositional, physical, and functional versatility [131,132,133]. For instance, Wang et al. [134] have synthesized a tumor-selective LPP consisting of a PEI/p21-saRNA-322 core and a hyaluronan-modulated lipid shell. The system was tested against colorectal cancer, and it was reported that it accumulated preferentially at the tumor site, leading to superior antitumor efficacy in vitro and in vivo. Alternatively, Shah et al. [135] have proposed the incorporation of ultrasound contrast agents in the liposomal cavity, followed by polyplexes addition. Thus, the scientists obtained ultrasound-activated LPPs that promote cancer cell uptake, elevate transfection efficiency, and reduce carrier cytotoxicity.
One more emerging delivery possibility is TNA transport via lipodendriplexes which are complex structures formed by non-covalent hybridization of dendriplexes with the liposomal membrane. In this respect, Tariq et al. [136] have incorporated pDNA-PAMAM-based dendriplexes into an optimized liposomal formulation (Figure 6). The researchers reported significantly improved pDNA transfection, lower LDH release, lower ROS generation, higher cellular protein content, and higher cell viability compared to dendriplexes without the lipid components. Thus, these new carriers can be considered promising candidates for efficient and biocompatible gene delivery systems.
To summarize the current status of lipid polymer-hybrid-based TNA carriers, Table 3 presents several examples of such delivery systems together with their potential applications.

5. Challenges and Emerging Solutions to Overcome Them

LNPs are undeniably promising innovative nonviral vectors for gene delivery. Nonetheless, several challenges still exist, limiting their potential. One of the main unresolved issues is represented by the poor endosomal escape after LNP cell entry. Attempting to understand this phenomenon, Herrera et al. [140] have created a highly sensitive and robust galectin 8-GFP (Gal8-GFP) cell reporter system to visualize the endosomal escape capabilities of LNP-encapsulated mRNA. This sensor system allows the rapid and efficient distinction of endosomal membrane integrity as an indicator of cytosolic availability of mRNA. Moreover, it helped the researchers identify differences in endosomal escape capabilities elicited by the varying sterol composition of mRNA LNP-based delivery systems.
Alternatively, Mihaila et al. [141] propose the optimization of siRNA LNP-mediated delivery by use of an ordinary differential equation (ODE)-based model. By means of mathematical modeling, the researchers designed and validated a predictive model that can compare the relative kinetics of different classes of LNPs towards choosing the best option.
Another problem associated with LNPs is that their intravenous administration results in liver accumulation where the reticuloendothelial system takes them up. To avoid this situation, Saunders et al. [142] propose the administration of a liposome (i.e., Nanoprimer) that can temporarily occupy liver cells as a pretreatment before LNPs delivery. The researchers obtained promising results as the Nanoprimer improved the bioavailability of tested RNA-encapsulated LNPs, increased protein production, and enhanced FVII silencing.
One more aspect that must be considered regarding lipid and polymer-based TNA delivery systems is the development of a biomolecular corona around these nanoparticles (Figure 7). This “crown” is formed by electrolytes, proteins, and lipids adsorbed on nanomaterials’ surfaces when in contact with a biological environment. As the association process is almost irreversible, the biomolecular corona defines the biological identity of the nanosystem, impacting in vivo characteristics, such as circulation time, biodistribution, uptake kinetics, macrophage recognition, release profile, or targeting [143,144,145].
Thus, when moving from in vitro models to in vivo studies or clinical trials, it may be expected to encounter a weaker delivery efficiency and therapeutic effect of carried nucleic acids. To overcome this issue, Yang et al. [147] have proposed the design of a corona made from cyclic RGDyK peptide-modified bovine serum albumin to be used as a precoat on a redox-responsive chitosan-based nanocarrier for siRNA delivery. The synthesized corona remained steady around the delivery vehicle, improved the system’s stability, biocompatibility, and targeting ability, reduced serum proteins adsorption, increased intracellular uptake, facilitated the lysosomal escape while maintaining the redox-sensitive responsiveness of the nanocarrier.

6. Conclusions

To conclude, TNAs represent a promising therapeutic strategy for a broad range of diseases. Benefiting from tremendous scientific interest in the recent years, numerous LNP-based delivery systems for pDNA, mRNA, siRNA, and other nucleic acids have appeared that can help fight against various cancers, infectious diseases, cardiovascular diseases, and inherited disorders. Several such formulations have reached the clinical trials testing stage or have even been approved for use in the general population in record time, as is the case of LNP-mRNA COVID-19 vaccines.
Other TNA delivery possibilities started to emerge as well, including polymer and lipid–polymer hybrid carriers. Nonetheless, these delivery systems are still in their infancy, most of them requiring further thorough research before moving from in vitro and in vivo tests to clinical studies.
Overall, the evolution in developing targeted and controlled release vehicles for efficient cellular uptake faces exponentially increasing progress. Given the recent research growth in the field and the precedent it has been created with the approval of the COVID-19 vaccines, it can be expected that other TNA delivery systems will soon enter the market.

Author Contributions

Conceptualization, A.-G.N., A.C.B. and A.M.G.; methodology, A.-G.N., A.C.B. and A.M.G.; writing—original draft preparation, A.-G.N., A.C.B. and A.M.G.; writing—review and editing, A.-G.N., A.C.B. and A.M.G. All authors have read and agreed to the published version of the manuscript.

Funding

The present work was possible due to the project 36355/23.05.2019 POCU/380/6/13.

Institutional Review Board Statement

Not applicable.

Acknowledgments

The present work was possible due to the project “Excelența academică și valori antreprenoriale—sistem de burse pentru asigurarea oportunităților de formare și dezvoltare a competențelor antreprenoriale ale doctoranzilor și postdoctoranzilor”—ANTREPRENORDOC (36355/23.05.2019 POCU/380/6/13).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Most important benchmarks in the development of lipid-based delivery systems for RNA. Adapted from [2,11,16,24], published by Nature Publishing Group, 2021; Elsevier, 2020; Nature Publishing Group, 2021; https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/376/366/safc-lipids-rna-wp7004en-ms.pdf, accessed on 10 October 2021.
Figure 1. Most important benchmarks in the development of lipid-based delivery systems for RNA. Adapted from [2,11,16,24], published by Nature Publishing Group, 2021; Elsevier, 2020; Nature Publishing Group, 2021; https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/376/366/safc-lipids-rna-wp7004en-ms.pdf, accessed on 10 October 2021.
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Figure 2. Visual representation of several conventional synthesis methods for LNP-based delivery systems. (a) hot high-pressure homogenization method; (b) cold high-pressure homogenization method; (c) solvent evaporation method; (d) microemulsion method. Adapted from [31], published by Uppsala University, 2020.
Figure 2. Visual representation of several conventional synthesis methods for LNP-based delivery systems. (a) hot high-pressure homogenization method; (b) cold high-pressure homogenization method; (c) solvent evaporation method; (d) microemulsion method. Adapted from [31], published by Uppsala University, 2020.
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Figure 3. Schematic representation of LNP-based delivery systems manufacturing through microfluidic methods. Adapted from [33], published by MDPI, 2020.
Figure 3. Schematic representation of LNP-based delivery systems manufacturing through microfluidic methods. Adapted from [33], published by MDPI, 2020.
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Figure 4. Cross-sectional model of SARS-CoV-2. Adapted from [60], published by AMER PUBLIC HEALTH ASSOC INC, 2021.
Figure 4. Cross-sectional model of SARS-CoV-2. Adapted from [60], published by AMER PUBLIC HEALTH ASSOC INC, 2021.
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Figure 5. Schematic representation of lipopolyplexes formation. Adapted from [131], published by Cell Press, 2019.
Figure 5. Schematic representation of lipopolyplexes formation. Adapted from [131], published by Cell Press, 2019.
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Figure 6. Schematic representation of lipodendriplexes formation and cellular internalization. (1) Endocytosis and pDNA release into the cytoplasm. (2) Transcription of gene-encoded DNA into mRNA. (3) mRNA export from the nucleus to the cytoplasm. (4) Protein expression. Adapted from [136], published by Nature, 2020.
Figure 6. Schematic representation of lipodendriplexes formation and cellular internalization. (1) Endocytosis and pDNA release into the cytoplasm. (2) Transcription of gene-encoded DNA into mRNA. (3) mRNA export from the nucleus to the cytoplasm. (4) Protein expression. Adapted from [136], published by Nature, 2020.
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Figure 7. (a) Initiation of formation of protein corona (PC) (seconds after the NP reaches the biological fluid); (b) beginning of exchange from the PC of proteins with low affinity with proteins that have higher affinity (seconds to minutes); (c) stabilized PC, with proteins with high affinity occupying the first layer of PC (hard PC) and the majority of the second layer (soft PC) where proteins with low affinity are still present. Adapted from [146], published by MDPI, 2020.
Figure 7. (a) Initiation of formation of protein corona (PC) (seconds after the NP reaches the biological fluid); (b) beginning of exchange from the PC of proteins with low affinity with proteins that have higher affinity (seconds to minutes); (c) stabilized PC, with proteins with high affinity occupying the first layer of PC (hard PC) and the majority of the second layer (soft PC) where proteins with low affinity are still present. Adapted from [146], published by MDPI, 2020.
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Table
Table 1. A summary of lipid-based nucleic acids delivery systems applications.
Table 1. A summary of lipid-based nucleic acids delivery systems applications.
Delivery SystemPhysicochemical PropertiesDisease/ConditionTesting StageAdministration RouteObservationsRef.
LNP-encapsulated C-24 alkyl phytosterols carrying mRNAShape: polyhedral
Hydrodynamic diameter: ~100 nm
Encapsulation efficiency: >90%
Lipid composition: ionizable lipid (DLin-MC3-DMA, Lipid 9, or DODMA):sterol:DSPC:DMG-PEG2k at molar ratios of 50:38.5:10:1.5		CancerIn vitro-High encapsulation efficiency
High cellular uptake and retention
Enhanced intracellular diffusivity
Enhanced gene transfection		[32]
Aerosolizable siRNA-encapsulated SLNsSize: 164.5 ± 28.3 nm
Polydispersity index: 0.31 ± 0.09
Zeta potential: −29.1 ± 8.6 mV
Lipid composition: lecithin, cholesterol, and lipid-PEG conjugate		Lung diseases
(e.g., cystic fibrosis)		In vitro-SLNs diffused through the simulated mucus layer
Possibility of down-regulating the expression of the gene of interest (e.g., TNF-α siRNA) by alveolar macrophages of lung epithelial cells
Promising for lung delivery by inhalation		[29]
mRNA encapsulated cationic lipid-modified aminoglycosides-based LNPsSize: 100–200 nm
Surface charge: ~0 mV
Encapsulation efficiency: >95%
Lipid composition: GT-EP10, DOPE, cholesterol, DMG-PEG2k		Liver diseasesIn vitro
and
In vivo (tested on mice)		IntravenousEfficient mRNA delivery to the liver
Safe delivery system (no obvious toxicity, changes in ALT or AST, or liver injury in histological sections)
Potential for application in gene editing and delivery of TNA		[85]
siRNA encapsulated LNPs with different surface chargesShape: spheres with filled cores
Size: ~7- nm
Zeta potential: ranged between 0 and 30 mV
Encapsulation efficiency: >99%
Lipid composition: lipidoid, DOTAP, DOPE, cholesterol, DSPE-PEG2k		Retinal diseases (e.g., age-related macular degeneration, glaucoma)In vitro
and
In vivo (tested on mice)		IntravitrealSuccessfully managed gene knockdown in mammalian cell line and primary neurons
No gene silencing facilitation in the retina pigmented epithelium layer
48 h after injection, neutral and mildly positive LNPs mediated limited retinal gene suppression (<10%), whereas more positive LNPs led to approx. 25% gene suppression in the retinal ganglion cell layer		[86]
GALA-modified LNP-encapsulated pDNASize: 125–155 nm
Zeta potential: ~15 mV (DOTMA-shell particles); ~−15 mV (YSK05-shell particles)
Composition: inner coat—DOPE; STR-R8; outer coat—DOTMA/YSK05, cholesterol		Lung diseasesIn vivo
(tested on mice)		IntravenousEfficient lung-selective delivery systemHigh gene expression level and significantly improved transfection activity in the lungs
Improved efficiency of gene expression at the intracellular level due to the double-coating		[87]
Formulated lipidoid nanoparticles (FLNP)-encapsulated modified mRNASize: ~155 nm
Lipid composition: epoxide-derived lipidoid complex (C14-113)		Cardiovascular diseaseIn vivo (tested on rats and pigs)Intramyocardial injectionHighly efficient, rapid, and short-term mRNA expression in the heart
Over 60-fold higher mRNA levels in heart tissue than for naked TNA
Limited off-target biodistribution		[88]
LNP-encapsulated mRNA encoding short-lived factor VIII (FVIII) proteinSize: <100 nm
RNA encapsulation: >80%
Lipid composition: ionizable lipid:DSPC:cholesterol:PEG lipid, at molar ratios of 50:10:38.5:1.5		Hemophilia AIn vivo
(tested on mice)		IntravenousSafe and effective delivery platform
Produced rapid and prolonged duration of FVIII expression
Easy to improve FVIII function by modification of mRNA sequence encoding newly developed FVIII protein with higher activity or longer half-lifeIt could be applied to prophylactic treatment and potentially various other treatment options		[89]
Ultra-small LNPs encapsulating sorafenib and midkine-siRNASize: 60.47 ± 6.9 nm
Zeta potential: −17.4 ± 5 mV
Sorafenib encapsulation efficiency: 96.5 ± 4.8%
RNA encapsulation efficiency: 94.5 ± 6.5%
Lipid composition: YSK05:DOPE:cholesterol:PEG-SP94:PEG, at molar ratios of 5:2:3:1:0.3		Sorafenib-resistant hepatocellular carcinomaIn vivo
(tested on mice)		IntravenousEnhanced tumor accumulation, selectivity, and in vivo gene silencing
Biosafe treatment approach
Synergistic action allowed the eradication of hepatocellular carcinoma at a low drug dose (2.5 mg/kg), which is unattainable with individual monotherapy		[90]
LNP-encapsulated SARS-CoV-2 human Fc-conjugated receptor-binding domain mRNASize: ~100 nm
Polydispersity index: <0.3
Lipid composition: ionizable lipid, DSPC, DMG-PEG, cholesterol		COVID-19In vivo
(tested on mice)		IntramuscularCell-free, simple, and rapid vaccine platform
Produced robust humoral response
Lead to a high level of neutralizing antibodies and a Th1-biased cellular response
It could be used for LNP-based mRNA vaccines in general and for a COVID19 vaccine in particular		[91]
HIV-1 Env-encoded as nucleoside-modified mRNA-LNPSize: ~80 nm
Lipid composition: ionizable cationic lipid, phosphatidylcholine, cholesterol, PEG-lipid		HIV infectionIn vivo (tested on rhesus macaques)IntramuscularElicited durable neutralizing antibodies that were stable for at least 41 weeks
Equal or better immune response when compared to adjuvanted recombinant protein vaccines		[81]
LNP-encapsulated HSV-2 nucleoside-modified mRNAn.r.HSV-2 infectionHSV-1 infectionIn vivo (tested on mice)IntramuscularPotent protection against HSV-1 and HSV-2 genital infections
Better at limiting virus replication in the genital tract than protein-based vaccine alternative
Prevented HSV invasion of the dorsal root ganglia in 97.5% of infected mice		[82]
Atu027 (liposome-encapsulated siRNA)Composition: positively charged AtuFect01, a neutral, fusogenic DPhyPE helperlipid and the PEGylated lipid MPEG-2000-DSPE, at molar ratios of 50:49:1Advanced or metastatic pancreatic adenocarcinomaClinical trial—Phase 1/2IntravenousUsed in combination with gentamicin-based chemotherapeutic treatment
Enhanced antitumor activity
Disease stabilization for 41% of patients		[92,93,94]
MTL-CEBPA
(double-stranded RNA formulated into a SMARTICLES® liposomal nanoparticle)		Lipid composition: liposomal formulationAdvanced hepatocellular carcinoma
Cirrhosis		Clinical trial—Phase 1IntravenousAdministered as monotherapy or in combination with sorafenib
Acceptable safety profile (comparable to drugs such as sorafenib, regorafenib, and nivolumab)Antitumor activity with a mean progression-free survival of 4.6 months		[95,96]
EphA2-targeting DOPC-encapsulated siRNALipid composition: DOPCAdvanced/recurrent malignant solid neoplasmClinical trial—Phase 1IntravenousDose-escalation study
Potential of slowing tumor cells growth by shutting down the causative gene		[97]
DCR-MYC (synthetic double-stranded RNA in a stable LNP suspension)n.r.Solid tumors
Multiple myeloma
Non-Hodgkins Lymphoma
Pancreatic neuroendocrine tumors
Primitive neuro-ectodermal tumors		Clinical trial—Phase 1IntravenousDose-escalation study
Antitumor potential by inhibiting MYC oncogene and thereby inhibiting cancer cell growth		[98]
mRNA-2416 (LNP-encapsulated mRNA encoding human OX40L)n.r.Relapsed/refractory solid tumor malignancies or lymphoma
Ovarian cancer		Clinical trial—Phase 1/2IntratumoralDose-escalation study
Administered alone or in combination with fixed doses of durvalumab
Potential immunomodulatory and antitumor activities		[99]
SGT-53 (cationic liposome encapsulating a normal human wild type p53 DNA sequence in a plasmid backbone)Lipid composition: complex DOTAP:DOPE cationic liposomeNeoplasm
Recurrent/refractory solid tumors in pediatric patients		Clinical trial—Phase 1IntravenousDose-escalating study
Administered alone or in combination with topotecan and cyclophosphamide
Potential to significantly sensitize pediatric cancer cell lines to the killing effect of the standard chemotherapeutic agents		[100,101]
SGT-53Lipid composition: complex DOTAP:DOPE cationic liposomeMetastatic pancreatic cancerClinical trial—Phase 2IntravenousAdministered in combination with gemcitabine/nab-paclitaxel
Efficient and specific delivery of p53 cDNA to tumor cells
Expected to restore wtp53 function in the apoptotic pathway		[101,102]
JVRS-100
(cationic liposome-plasmid DNA complex)		Lipid composition: DOTIM/cholesterol cationic liposomeRelapsed/refractory leukemiaClinical trial—Phase 1IntravenousDose-escalation study
Accelerated titration schema followed with one patient at each dose levelPotential immunostimulant activity		[103]
mRNA-1273 (LNP-encapsulated mRNA encoding for full-length perfusion stabilized spike protein of SARS-CoV-2)Lipid composition: ionizable lipid SM-102, DSPC, cholesterol, DMG-PEG2k, at molar ratios of 50:10:38.5:1.5COVID-19Clinical trial—Phase 1IntramuscularDose-ranging studyEvaluation of the safety and reactogenicity of a second dose vaccination schedule[104,105]
mRNA-1273Lipid composition: ionizable lipid SM-102, DSPC, cholesterol, DMG-PEG2k, at molar ratios of 50:10:38.5:1.5COVID-19Clinical trial—Phase 2IntramuscularAnalyze the development of cellular and humoral immunity against SARS-CoV-2 after administration of the third dose of vaccine in renal or renopancreatic transplant patients who have remained seronegative after the standard two-dose regimen[105,106]
LNP-encapsulated mRNA encoding the receptor-binding domain (RBD) of spike glycoprotein of SARS-CoV-2Lipid composition: lipid 9001, cholesterol, DSPC, DMG-PEG2kCOVID-19Clinical trial—Phase 3IntramuscularEvaluation of humoral immunity induced by the investigational vaccine and solicited adverse effects observed within 7 days post-immunization
Evaluation of protective efficacy
Adverse events collection over 0–28 days after each vaccination
Serious adverse events collection from Dose 1 through 12 months post complete series		[107]
Comirnaty (COVID-19 mRNA-embedded in LNPs)Lipid composition: ALC-0315, DSPC, cholesterol, ALC-0159, at molar ratios of 46.3:9.4:42.7:1.6COVID-19Clinical trial—Phase 4IntramuscularVaccine given in two doses in patients with primary or secondary immunosuppressive disorders
Results compared to a group of healthy control individuals
Investigation of safety and immune responses under 6 months of time for each immunized participant		[105,108]
BNT163b2 (LNP-formulated nucleoside-modified RNA encoding for full-length perfusion stabilized spike protein of SARS-CoV-2)Lipid composition: ALC-0315, DSPC, cholesterol, ALC-0159, at molar ratios of 46.3:9.4:42.7:1.6COVID-19Clinical trials—Phase 4IntramuscularAdministration of a third vaccine dose in adults who received two doses of an inactivated COVID-19 vaccine at least 3 months prior to the study
Evaluation of humoral immunogenicity, reactogenicity, and safety of a heterologous third vaccination dose		[109]
mRNA-1273.351 (LNP-encapsulated mRNA encoding for full-length perfusion stabilized spike protein of SARS-CoV-2 B.1.351 variant)Lipid composition: ionizable lipid SM-102, DSPC, cholesterol, DMG-PEG2k, at molar ratios of 50:10:38.5:1.5COVID-19Clinical trial—Phase 1IntramuscularEvaluation of the safety, reactogenicity, and immunogenicity of the new vaccine variant
Investigations on both naïve and previously vaccinated individuals		[110]
n.r.—not reported.
Table
Table 2. A summary of polymer-based nucleic acids delivery systems applications.
Table 2. A summary of polymer-based nucleic acids delivery systems applications.
Delivery SystemPhysicochemical PropertiesDisease/ConditionTesting StageAdministration RouteObservationsRef.
Modified dendrimer nanoparticle (MDNP)-based RNA repliconComposition: modified PAMAM dendrimer:DMPE-PEG2k:RNA at a mass ratio of 11.5:1:2.3Zika virus infectionEx vivo (tested on mice)-Tool to generate ZIKV vaccine in the absence of reference virus stocks
Elicited ZIKV E protein-specific IgG responses
Assessment of immune responses without the need for recombinant production of native glycoprotein		[118,119]
Ab-conjugated LGA-PEI NPs for the delivery of TNAsSize: ~100–200 nm
Composition: LGA, PEI, TNAs in various mass ratios		Pancreatic cancerIn vitro
and
In vivo (tested on mice)		IntravenousEffective loading of TNAs, such as pDNA, mRNA, and miRNA, resulting in stable and functionalized nucleic acids
Improved binding, internalization, and gene expression in pancreatic cancer cells with high expression of targeted cell surface markers
The developed systems can be extended to other types of cancers that express specific biomarkers		[115]
STAT3-targeting PEI-encapsulated siRNASize: 135.1 ± 5.2 nm
Zeta potential: 34.6 ± 2.2. mV
N/P ratio: 10		Skin melanomaIn vitro
and
In vivo (tested on mice)		IntradermalMinimally invasive administration
Enhanced cellular uptake and transfection of siRNA
Enhanced gene silencing efficiency
Inhibited tumor cells growth in a dose-dependent manner		[117]
PEI-based nanoparticle encapsulated with IGF1 modified mRNAN/P ratio: 6Hypoxia
Myocardial infarction		In vivo (tested on mice)Intramyocardial injectionPotential for an extended cytoprotective effect of transient IGF1
Promoted cardiomyocyte survival and abrogated cell apoptosis under hypoxia-induced apoptosis conditions
Induced downstream increases in the levels of Akt and Erk phosphorylation		[120]
CALAA-01 (cyclodextrin containing polymer encapsulating anti-R2 siRNA)Composition: duplex of synthetic, non-chemically-modified siRNA (C05C), cyclodextrin-containing polymer (CAL101), stabilizing agent (AD-PEG), targeting agent (AD-PEG-Tf)Relapsed or refractory cancer
Solid tumors		Clical trial—Phase 1IntravenoussiRNA-containing nanocomplexes are targeted to cells that overexpress the transferrin receptor (TfR)
transferrin binds to TfRs on the cell surface, and the siRNA-containing nanocomplex enters the cell by endocytosis
inside the cell, the polymer unpacks the siRNA, allowing it to function via RNA interference
significant intratumoral downregulating of the target protein		[121]
siG12D-LODERComposition: miniature biodegradable biopolymeric matrix loaded with siRNAPancreatic ductal adenocarcinomaPancreatic cancerClinical trial—Phase 2ImplantationHighly effective and safe device implantation
High safety and tolerability profiles
Progression-free survival in the study population		[122]
Table
Table 3. A summary of lipid-polymer hybrid-based nucleic acids delivery systems applications.
Table 3. A summary of lipid-polymer hybrid-based nucleic acids delivery systems applications.
Delivery SystemPhysicochemical PropertiesDisease/ConditionTesting StageAdministration RouteObservationsRef.
PEGylated CL4H6-MRTF-B siRNA-loaded LNPsSize: ~200 nm
Shape: spherical
N/P ratio: 7.5
Composition: CL4HS:DOPE: PEG-DMG. At molar ratio of 50:50:1		Conjunctival fibrosisIn vitro-Non-toxic at a concentration of 50 nM
Effectively silenced the MRTF-B gene
Enhanced encapsulation efficiency
Decreased fibroblast contraction after a single transfection
Remarkable efficiency even after long periods of refrigeration		[123]
Ultrasound-activated LPPsSize: ~170–250 nmComposition: polyplexes—PEI, pDNA; lipid formulation—DPPC, cholesterol, DPPG, PEG40SOvarian cancerIn vitro-Safe method for gene delivery
Enhanced transfection efficiency and low cytotoxicity when exposed to low-frequency ultrasound
Ultrasound exposure at a specific post-transfection time interval promotes carrier’s entry through the ECM of the cells into an intracellular environment
Intracellular uptake is reported even in the presence of chlorpromazine, which seems to be the carrier’s endosomal escape		[135]
DLNPs containing PEGylated BODIPY dyesSize: ~138 nm
Zeta potential: ~−1.0 mV
Composition: dendrimer-based lipid nanoparticle with BODIPY core, indole linker, and PEG-lipid of lengths between 1000 and 2000 g/mol		CancerIn vitro
and
In vivo
(tested on mice)		IntravenousParticles formulated with a pH-responsive PBD-lipid produced 5—to 35-fold more functional protein than control ones formulated with traditional PEG-lipid in vitro
Enhanced mRNA delivery potency in vivo
Efficient functional protein expression
Successfully mediated mRNA expression in tumors and simultaneously illuminated tumors via pH-responsive NIR imaging		[125]
PEGylated ionizable LNPs formulated with mRNASize: 60–100 nm
Composition: DOPE, cholesterol, C16-PEG2000 ceramide
Ionizable lipids:mRNA ratio: 10:1		Chronic liver diseases (e.g., liver fibrosis, cirrhosis)In vitro
and
In vivo (tested on mice)		IntravenousParticles obtained through microfluidic synthesis
Particles were mostly distributed to the liver
The highest transfection efficiency among different types of liver cells was reported for hepatocytes		[137]
siRNA-loaded LNPs conjugated with a PEG-monacyl fatty acidSize: ~50–90 nm
Zeta potential: ~ -4.3 - + 4.3 mV
Composition: YSK05, DSPC, cholesterol, PEG-DMG, PEG-MO		CancerIn vitro
and
In vivo (tested on mice)		Intraperitoneal injectionSignificantly improved siRNA delivery efficiency as compared to originally developed LNPs (i.e., [138])
siRNA was stably retained in mouse serum, leading to a gene knockdown effect
Significant gene silencing in cancer cells, ascites, and solid tumor of the mesentery		[139]
PLGA-LNPs loaded with CRISPR/Cas9 plasmidsSize: 179.6 ± 44.82 nm
Zeta potential: 29.6 ± 4.33 mV
Encapsulation efficiency: 76.5 ± 7.2%
Composition: PLGA, lecithin, DSPE-PEG-cRGD, DSPE-PEG-biotin (lipids at a molar ratio of 7:1.5:1.5)		GlioblastomaIn vitro
and
In vivo (tested on mice)		IntravenousEffective gene delivery
Restored sensitivity of glioblastoma cells to temozolomide (TMZ)
Highly safe and biocompatible multi-functional system
Potential treatment for TMZ-resistant glioblastoma and TNA delivery to the central nervous system		[130]
Tumor-selective LPP-p21-saRNA-322Size: 230.2 ± 10.3 nm
Polydispersity index: 0.15 ± 0.02
Zeta potential: −17.3 ± 0.4 mV
Composition: HA-PE tumor-selective liposomes, PEI-p21-saRNA-322 polyplexes		Colorectal cancerIn vitro
and
In vivo
(tested on mice)		RectalHigh drug accumulation in the tumor site
Efficient intracellular delivery and lysosome escapement
Significant inhibition of orthotopic colorectal tumor growth
Biocompatible delivery system; growth inhibition effect attributed to p21-saRNA-322 rather than the carrier		[134]
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Niculescu, A.-G.; Bîrcă, A.C.; Grumezescu, A.M. New Applications of Lipid and Polymer-Based Nanoparticles for Nucleic Acids Delivery. Pharmaceutics 2021, 13, 2053. https://doi.org/10.3390/pharmaceutics13122053

AMA Style

Niculescu A-G, Bîrcă AC, Grumezescu AM. New Applications of Lipid and Polymer-Based Nanoparticles for Nucleic Acids Delivery. Pharmaceutics. 2021; 13(12):2053. https://doi.org/10.3390/pharmaceutics13122053

Chicago/Turabian Style

Niculescu, Adelina-Gabriela, Alexandra Cătălina Bîrcă, and Alexandru Mihai Grumezescu. 2021. "New Applications of Lipid and Polymer-Based Nanoparticles for Nucleic Acids Delivery" Pharmaceutics 13, no. 12: 2053. https://doi.org/10.3390/pharmaceutics13122053

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