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Recent progress on nanosystems for nucleic acid delivery

RSC Pharmaceutics

August 2024
1(4):645-674

DOI:10.1039/D4PM00009A

License
CC BY 3.0

Authors:

Shanka Walia

Indian Institute of Technology Gandhinagar

Mohit J Mehta

Tampere University

Nucleic acid (NA) based therapeutics have witnessed tremendous progress and breakthroughs in treating pathological conditions, including viral infections, neurological disorders, genetic diseases, and metabolic disorders. NAs such as plasmid DNA (pDNA), short interfering RNA (siRNA), microRNA (miRNA), and antisense oligonucleotides (ASOs) can be modified to revolutionize personalized medicine. Despite the great potential of NA-based therapeutics, their clinical transformation is significantly hampered by instability, degradation, and inefficient delivery to the targeted site in the in vivo system. Lipid-based delivery systems hold great potential to overcome these shortcomings to enhance the delivery and bioavailability, improve stability, and increase the therapeutic effect of the NAs by delivering them to the active site. This review emphasized various nucleic acid-based therapeutics and their enhanced and improved delivery using different nanocarriers. Ultimately, the importance of lipid-based nanocarriers for delivering NAs is discussed and provides perspective in this field.

(a) Impact of AuNP:structural RNA complex on MDA-MB-231 GFP/RFP cells. In vitro studies show the regulation of CopGFP expression in the targeted cells. Adapted with permission from ref. 97. Copyright 2021 ACS Chemical Society. (b) Confocal microscopy images of cells incubated with (A–C) nanoparticle dimers and (D−F) “non-targeting” scrambled nanoparticle dimers. Sixteen HBE cells express only keratin 8, and MRC 5 cells express only vimentin. A549 expresses both keratin 8 and vimentin. A fluorescence signal corresponding to the presence of keratin 8 mRNA (A), vimentin mRNA (B), and both vimentin and keratin 8 mRNA (C) are observed. When incubated with nanoparticle dimers designed with “non-targeting” scramble sequences, all three cell lines display no response. Adapted with permission from ref. 98. Copyright 2018 ACS Chemical Society

…

(a) Schematic illustration of QD-NBs for HIV-1 genomic RNA detection. (A) Schematic diagram of the QD-NBs’ preparation; schematic illustration of QD-NBs for (B) HIV-1 genomic RNA detection in living cells, and (C) fluorescence labeling of single virus particles. Adapted with permission from ref. 108. Copyright 2019 American Chemical Society. (b) Reaction scheme for the chemical functionalization of SiNPs with histidine and lysine and the complexation of pDNA. Adapted with permission from ref. 114. Copyright 2019 Royal Society of Chemistry. (c) Confocal laser scanning microscopic images of MGC-803 cells incubated with Cdots@PEI or Cy3-siRNA for 2 h, or Cy3-siRNA-Cdots@PEI complexes for 2 and 5 h. Adapted with permission from ref. 117. Copyright 2014 Springer Nature. (d) GFP silencing by PEI-coated SPMNs/GFP shRNA in GFP-transfected 3D cell cultures. Adapted with permission from ref. 123. Copyright 2010 American Chemical Society

…

(a) Phase contrast microscopic images of control and antisense miR-21 transfected cells treated with 500 μM TMZ. In vitro cell viability MTT assays on control and antisense miR-21 transfected (b) U87 MG cells, (c) LN 229 cells, and (d) T98G cells treated with different concentrations of TMZ. (e) Cellular pathway analysis of antisense miR-21 and TMZ co-treatment on U87 MG cells. Adapted with permission from ref. 131. Copyright 2015 American Chemical Society. (f) Schematic representation of PAMAM dendrimer/pDNA-p53 nanocarrier preparation for cancer gene therapy. Adapted with permission from ref. 139. Copyright 2021, American Chemical Society

…

FDA-approved lipid-based structures contain some variation of the four essential components: cholesterol, a helper lipid, a PEG-lipid, and a cationic or ionizable lipid. (a) Lipid-based structures can include micelles, which consist of a lipid monolayer, or liposomes, which consist of a bilayer. Lipid nanoparticles comprise multiple lipid layers and lipid and nucleic acid microdomains. (b) and (c) In addition to the RNA payload, LNPs often consist of cholesterol, a helper lipid, a PEG-lipid (all shown in part b), and a cationic or ionizable lipid (part c). (d) The molar ratios of the four components constitute the FDA-approved Acuitas/BioNTech/Pfizer COVID-19 vaccine and patisiran, which delivers siRNA to the liver. Adapted with permission from ref. 195. Copyright 2022, Springer Nature

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(a) Preparation of LNPs derived from ionizable lipids. (b) In vivo evaluation of ILs showed potent luciferase expression. Ex vivo organ images showed that LNPs were mostly taken up into the liver. Liver histology image and transfection efficiency showed significant tdTomato fluorescence in the hepatocyte. cv, central vein. Adapted with permission from ref. 220. Copyright 2021, American Association for the Advancement of Science. (b) Synthesis and in vivo application of CP liposomes. (c) Tumor therapy efficacy of CP liposomes (DDCP-c, DMCP-c, DPCP-c, and folate-CP) with DPPC as the control. Adapted with permission from ref. 228. Copyright 2021, American Chemical Society

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Cite this: RSC Pharm., 2024, 1,645

Received 16th January 2024,

Accepted 5th August 2024

DOI: 10.1039/d4pm00009a

rsc.li/RSCPharma

Recent progress on nanosystems for nucleic acid

delivery

Shanka Walia

and Mohit J. Mehta *

b,c,d

Nucleic acid (NA) based therapeutics have witnessed tremendous progress and breakthroughs in treating

pathological conditions, including viral infections, neurological disorders, genetic diseases, and metabolic

disorders. NAs such as plasmid DNA (pDNA), short interfering RNA (siRNA), microRNA (miRNA), and anti-

sense oligonucleotides (ASOs) can be modiﬁed to revolutionize personalized medicine. Despite the great

potential of NA-based therapeutics, their clinical transformation is signiﬁcantly hampered by instability,

degradation, and ineﬃcient delivery to the targeted site in the in vivo system. Lipid-based delivery systems

hold great potential to overcome these shortcomings to enhance the delivery and bioavailability, improve

stability, and increase the therapeutic eﬀect of the NAs by delivering them to the active site. This review

emphasized various nucleic acid-based therapeutics and their enhanced and improved delivery using

diﬀerent nanocarriers. Ultimately, the importance of lipid-based nanocarriers for delivering NAs is dis-

cussed and provides perspective in this ﬁeld.

1. Introduction

Nucleic acids (NAs) are immensely recognized for their thera-

peutic potential during the COVID-19 pandemic. NAs, includ-

ing DNA, antisense oligonucleotides (ASO), aptamers, short

interfering RNA (siRNA), microRNA (miRNA), and messenger

RNA (mRNA), provide a highly versatile platform at the under-

lying level of transcription and translation for the treatment

of diseases as compared with traditional drugs.

1,2

Nucleic

acids’high specificity and pharmacological eﬀect oﬀered un-

precedented opportunities to combat viral infections and

complicated diseases, including cancer, diabetes, cardio-

vascular diseases, genetic disorders, and acquired con-

ditions.

The majority of the NAs bind selectively to their

target molecules via complementary Watson–Crick base

pairing, making target molecule detection simple.

NAs are

good candidates for the personalized medicinal approach

because genes and their mutations can be easily questioned.

Hence, NAs can target patient-specific genes with minimal

oﬀ-target eﬀects.

In contrast, pharmaceutical drug mole-

cules require extensive screening and medicinal chemical

optimization.

However, the intracellular uptake of NAs is quite challen-

ging because of their anionic nature and poor stability, which

makes the intracellular uptake of NAs diﬃcult. Due to their

highly hydrophilic and polyvalent anionic properties, NAs are

degraded by extracellular enzymes and, thus, poorly absorbed

by cells. When injected intravenously, they tend to accumulate

in the liver and kidneys.

All these factors reduced the

eﬃciency of the delivered cargo to elicit the desired response.

Furthermore, due to their high molecular weight, negative zeta

potential, and hydrophilicity, NAs cannot penetrate the cellular

membrane. However, clinical application is the primary goal

of any therapeutic molecule; hence, nanovehicles should

ensure adequate delivery of drugs to the targeted cell or organ

without causing any harmful eﬀect on the healthy cells. The

therapeutic payload is delivered to the targeted cells via two

main approaches, namely, passive targeting and active target-

ing. The foundation of passive targeting is the enhanced per-

meability and retention (EPR) eﬀect. For example, tumor vas-

culature is often leaky compared with healthy blood vessels.

This helps nanoparticles to penetrate the cancer cells more

eﬃciently.

In contrast to this, active targeting facilitates

surface modifications of nanovehicles with specific ligands,

resulting in enhanced binding to the receptors of targeted

cells. Presently, a structure capable of improving the bio-

eﬃcacy and controlled release of NAs at the desired site is of

the utmost importance. Nucleic acids possess versatile

physicochemical properties and tunability. These features

allow them to be easily functionalized with various bio-

Swami Sahajanand College of Computer Science, Plot No. 639, ISCON Mega City,

Gujarat, 364002, India

Nanomedicine Lab, Department of Biological Sciences and Bioengineering, Inha

University, 100 Inha-ro, Michuhol-gu, Incheon, 22212, Republic of Korea

Adult Stem Cell Group, Faculty of Medicine and Health Technology, Tampere

University, Tampere, 33520, Finland

Tays Research Services, Wellbeing Services County of Pirkanmaa, Tampere

University Hospital, Tampere, 33520, Finland. E-mail: mohitjmehta@gmail.com,

mohit.mehta@tuni.fi

molecules and nanoparticles (NPs) for enhanced therapeutic

eﬀect with selective binding to the targeted molecules.

Diﬀerent nanoparticle-based platforms such as metal NPs,

polymers, dendrimers, proteins, lipids, and other bio-

molecules are widely exploited in conjugation with NAs.

9,10

For

instance, liposomes and micelles, which have now received

FDA approval, were part of the first generation of nanoparticle-

based therapy.

Lipid-based nanocarriers were introduced as

promising tools to retain the structural integrity and thera-

peutic potential of NAs. Lipid-based nanocarriers oﬀer a versa-

tile platform for NA encapsulation, which resulted in the clini-

cal translation of several NA therapeutics. This review aims to

provide an in-depth discussion on various nanosystems for

nucleic acid delivery and therapeutics. They oﬀer a powerful

approach to address the limitations of traditional treatments,

and hence can potentially overcome these challenges.

2. Nucleic acid-based therapeutics

In the last decade, nucleic acid-based therapies have emerged

as promising approaches for treating multiple disorders by

regulating molecular pathways.

NAs target disease-causing

genes in a specific manner, enabling precise and personalized

treatment for life-threatening diseases.

NA-based thera-

peutics knock down, upregulate, or alter targeted gene

expression. Currently, several NA-based therapeutics are used

for the treatment of severe ailments (Table 1).

2.1 Plasmids

Plasmids are circular, double-stranded, high molecular weight

(<1000 to >200 000 bp) DNA constructs. They are often used as

vectors to introduce a foreign gene into a target cell for various

purposes such as gene therapy, vaccine production, and

protein expression.

14,15

The plasmid backbone contains the

gene for antibiotic resistance controlled by a prokaryotic pro-

moter (a prokaryotic origin of replication for plasmid replica-

tion),

and an expression cassette with a promoter to initiate

the transcription of a transgene that encodes a therapeutic

protein.

Plasmid DNA molecules typically contain several

regulatory signals, such as promoter and enhancer sequences,

responsible for regulating gene expression.

Promoters (cyto-

megalovirus (CMV) and Rous sarcoma virus or human alpha-

actin, human beta-actin promoter) commence gene transcrip-

tion by providing a recognition site for the RNA polymerase.

Enhancers play a vital role in the large-scale production of a

gene with specificity. They are localized either downstream or

upstream from the promoter site. Transcription eﬃciency can

be elevated by selecting the promoter and enhancer of interest;

for example incorporating SV40 enhancer in the expression

plasmid enhanced muscle-specific gene expression.

Plasmids also play an essential role in generating vaccines for

genetic immunization. Plasmid-derived vaccines are easy to

produce if a genetic sequence of the concerned variant is

identified, and are also known to control the infection.

Pardridge et al. (2020) developed a plasmid DNA approach for

the treatment of Niemann–Pick C1 (NPC1), a lysosomal chole-

sterol storage disorder aﬀecting the brain.

Plasmid DNA was

encapsulated in pegylated liposomes encoding the functional

human NPC1 gene. Quantitative PCR has shown the successful

delivery of pDNA and NPC1 mRNA expression in the brain,

liver, and spleen. Malardo and coworkers (2012) demonstrated

that injecting a low dose of pDNA (pcDNA3) in the Wistar rat

endotoxemia model has increased plasma vasopressin and

regulates blood pressure.

The rats treated with 10–20 µg

pDNA showed lower levels of inflammatory cytokines, namely,

IL-6 and TNF-α.

2.2 Antisense oligonucleotide (ASO)

Antisense oligonucleotides (ASOs) of therapeutic importance

comprise single-stranded nucleic acids and are 18–30 base

pairs in length.

ASOs form complementary base pairs with

their target RNA by classical Watson and Crick base pairing.

After binding with the target RNA, ASOs resulted in gene silen-

cing by altering or degrading the expression of the target RNA

via cleavage or blockage.

16,17

The various mechanisms adopted

by ASOs to degrade target RNA include (1) modified splicing

by skipping and inclusion of exons, and inhibition of 5′cap

formation, (2) steric blockage of ribosomal functions as they

inhibit the binding of target RNA with ribosomes through the

translation arrest of the target RNA, and (3) induction of

RNAse H that recognizes ASO–RNA hybrids and degrade the

target RNA present in the hybrid.

31,32

ASOs are primarily admi-

nistered via transfection and transduction in vitro and

in vivo.

For example, Passini et al. (2011) used 2′-O-2-methox-

yethyl-modified ASOs for the treatment of severe spinal muscu-

lar atrophy (SMA).

SMA is an autosomal recessive neuromus-

cular disorder related to the deficiency of survival motor

neuron (SMN) protein caused by mutations in the SMN1 gene.

ASO injection resulted in increased levels of SMN protein in

Table 1 Applications of diﬀerent types of nucleic acid in clinical research

S. no. Nucleic acid Biomedical application Ref.

1 Plasmid DNA Gene cloning, gene therapy, DNA vaccine, protein expression, gene editing 14 and 15

2 Antisense oligonucleotides (ASOs) Targeting RNA functioning of genetic disorders, gene silencing 16 and 17

3 Aptamers Cell tracking, bacterial and viral protein sensing 18

4 siRNA and miRNA Gene therapy, antiviral therapy, drug discovery 19–21

Biomarkers, cancer treatment

5 mRNA Vaccine production, cancer immunotherapy, gene editing 22 and 23

6 CpG DNA Immunotherapy, vaccine adjuvant, gene therapy, anti-inflammatory 24 and 25

Review RSC Pharmaceutics

SMA-infected mice. In vivo studies suggested improved thera-

peutic eﬀects on muscle physiology, functioning, and the sur-

vival rate of motor neurons. Some examples of FDA-approved

ASO-based drugs include eteplirsen, golodirsen, and nusiner-

sen.

Several others are under clinical trials for the treatment

of neurological disorders, hepatitis B virus infections, solid

tumors, and renal diseases.

Although ASOs might have shown enhanced eﬃcacy in vitro

and in vivo, they showed drawbacks related to low cellular

uptake and poor stability in body fluids that hindered their

potential from bench to bedside.

2.3 Aptamers

Aptamers are short single-stranded synthetic oligonucleotides

of 10–100 nucleotides folded into three-dimensional

shapes.

36,37

Aptamers possess a remarkable tendency to bind

non-covalently to their target with high selectivity and speci-

ficity. Aptamers are widely explored to target biomolecules of

interest, including proteins, peptides, carbohydrates, anti-

bodies, small molecules, toxins, live cells, and even heavy

metals.

Thus, aptamers are potential candidates for cell

tracking, bacterial and viral protein sensing, medicine, and

analytical chemistry. Aptamers are primarily generated in vitro

from the systematic evolution of ligands by exponential enrich-

ment (SELEX).

Aptamers are often compared with antibodies

because of their similar function in binding the proteins with

specificity. Unfortunately, antibodies are fraught with signifi-

cant shortcomings, such as limited and unreproducible syn-

thesis, being expensive, and being prone to generating immu-

nogenicity. However, aptamers with versatile properties have

gained much attention in the research community since their

discovery in 1990.

39,40

Aptamers are a fascinating alternative to

conventional antibodies due to their facile and reproducible

production, small size, reduced immunogenic eﬀect, physico-

chemical stability, longer shelf life, and in vitro chemical

synthesis.

2.4 Small interfering RNA and microRNA

Small interfering RNA (siRNA) and microRNA (miRNAs) are

representative modulators of the interference RNA (RNAi)

mechanism. RNAi comprises a group of agents that use

double-stranded RNA containing homologous sequences

complementary to the target gene and perform sequence-

specific gene silencing.

The primary function of RNAi is to

build a robust defense mechanism for protecting the genome

from mobile genetic material released from viruses, which, on

activation, produce abnormal dsRNA or RNA.

For example,

stable RNAi nanocomplexes with redox-sensitive glycol chito-

san derivatives were synthesized based on rolling circle tran-

scription. This nanocomplex ensured systemic and targeted

siRNA delivery with enhanced therapeutic eﬀects in vivo via

the EPR eﬀect.

A nanomedicine platform based on AuNPs co-

valently functionalized with siRNA duplexes was eﬃciently

used to neutralize oncogene expression in glioblastoma multi-

forme.

In vivo studies indicated that the AuNP–siRNA nano-

complex penetrates the blood–brain barrier. AuNP–siRNA

nanocomplex eﬀectively knocked down endogenous

Bcl2L12 mRNA and protein levels and sensitized glioma cells

toward therapy-induced apoptosis by targeting the oncoprotein

Bcl2L12. Elbashir and coworkers (2001), demonstrated that 21-

nucleotide siRNA duplexes mediated the knockdown of

endogenous and heterologous genes in human embryonic

kidney (293) and HeLa cells.

In 2018, the FDA approved the

first siRNA-based drug, patisiran (Onpattro), to treat familial

amyloid polyneuropathy.

Other siRNA-based medications

approved by the FDA for clinical use include givosiran (to treat

acute hepatic porphyria), lumasiran (for the treatment of

primary hyperoxaluria type 1), and inclisiran (to treat athero-

sclerotic cardiovascular disease).

Vir Biotechnology and

Alnylam Pharmaceuticals recently developed ALN-COV (VIR

2703) based on siRNA therapeutics to cure SARS-CoV and

SARS-CoV-2 infections.

microRNA (miRNA) is an attractive therapeutic tool, particu-

larly in cancer treatment. miRNA interacts with the 3′or 5′

untranslated region of the targeted mRNA, resulting in degra-

dation and translational suppression.

Li et al. (2021) reported

intracellular miRNA imaging and gene silencing using a let-7a

miRNA-activated DNA nanomachine.

Multifunctional

miRNA-515 sponge-loaded magnetic nanodroplets combined

with ultrasound and magnetism were used to treat hepatocel-

lular carcinoma (HCC).

In vivo, studies showed the suppres-

sion of xenograft HCC because miRNA-515 upregulated the

expression of anti-oncogenes, namely CD22, P21, TIMP1,

NFKB, and E-cadherin in cancerous cells. However, no miRNA

drug is available and approved by the FDA for therapeutic pur-

poses. Most of the miRNA drugs are still under clinical trials;

for example, miravirsen (miR-122 inhibitor) for treating hepa-

titis (Hep) C has completed Phase II clinical trials. Similarly,

lademirsen, or RG-012 (miR-21 inhibitor), is in Phase II clini-

cal trials for treating Alport syndrome (NCT02855268).

MRG-110 has completed phase I clinical trials, and further

studies are underway to treat impaired wounds. MRG-110

(miRNA-92a inhibitor) improved wound healing in preclinical

models and can eﬀectively treat impaired wound healing con-

ditions in diabetic patients. In short, several miRNA drugs are

under preclinical and clinical trials to treat various disorders.

2.5 Messenger RNA (mRNA)

Messenger RNA (mRNA) is a type of genetic material that con-

tains the information for producing proteins from DNA, the

genetic code found in the nucleus of a cell.

In recent years,

scientists have investigated the use of mRNA as a therapeutic

agent, specifically in the medical field. mRNA therapeutics,

commonly termed mRNA vaccines or mRNA drugs, are

designed from synthetic or modified mRNA molecules to

stimulate the production of specific proteins in the body to

treat diseases.

The Pfizer-BioNTech and Moderna COVID-19

vaccines, which employ mRNA to guide cells to generate the

spike protein present on the surface of the SARS-CoV-2 virus,

are among the most noteworthy milestones of mRNA thera-

pies.

The immune system then recognizes these spike pro-

teins as foreign moieties and stimulates an immune response

RSC Pharmaceutics Review

against the virus. This method has proved to be eﬃcient in

preventing critical illness and death from COVID-19, and regu-

latory agencies around the globe have permitted its emergency

usage.

51,52

Additionally, mRNA therapies can cure many ailments,

including cancer, genetic abnormalities, and viral

infections.

23,53

For instance, mRNA is used in cancer immu-

notherapy to engineer T cells and natural killer (NK) cells with

antigen receptors and as a template for immunologically active

proteins in various immune and non-immune cells.

mRNA

therapeutics also have the potential to treat genetic disorders

by providing cells with functional copies of genes that are

missing or non-functional. Researchers have already demon-

strated the ability to use mRNA to correct genetic mutations in

animal models of certain diseases, such as inherited retinal

diseases. They are working on advancing these therapies to

clinical trials.

mRNA therapeutics have several advantages

over traditional drugs. Because mRNA does not integrate into

the genome, it does not have the potential to cause permanent

genetic changes.

Moreover, mRNA can be easily synthesized,

thus allowing the rapid development and production of new

therapeutics. Despite these advantages, some challenges still

need to be overcome to fully realize the potential of mRNA

therapeutics. One of the main challenges is ensuring mRNA’s

safe and eﬀective delivery to the targeted cells. Researchers are

developing various delivery methods, such as NPs and viral

vectors, to overcome this challenge.

Furthermore, mRNA

therapeutics are relatively new, and more research is needed to

fully understand their safety and eﬃcacy.

Overall, mRNA

therapeutics represent a promising new approach to treating

and preventing various diseases and conditions. With contin-

ued research and development, mRNA therapeutics have the

potential to transform the way we think about medicine and

improve the lives of millions of people worldwide.

2.6 Cytosine-phosphate-guanine (CpG) DNA

Cytosine-phosphate-guanine (CpG) is a synthetic oligo-

nucleotide studied as a therapeutic agent for several undrug-

gable conditions. CpG activates the immune system by

binding to Toll-like receptor 9 (TLR9), expressed in immune

cells such as dendritic and B cells.

Consequently, these cells

are activated, and an immunological response is initiated.

Preclinical investigations demonstrated that CpG has anti-

tumor properties and enhances the immune response to

cancer cells, increasing tumor cell death.

24,58

It has been

reported that CpG has anti-inflammatory properties, making it

a suitable candidate for treating autoimmune illnesses such as

rheumatoid arthritis and multiple sclerosis.

CpG has been

shown to enhance the immune response to viral infections,

including influenza and herpes simplex virus, HIV,

60,61

hepa-

titis B and C,

62,63

and bacterial infections such as Streptococcus

pneumoniae.

25,64,65

In addition, CpG has been studied as a

potential treatment for allergies. In preclinical studies, CpG

has been found to reduce the severity of allergic reactions and

is currently being investigated as a treatment for allergies such

as asthma and allergic rhinitis.

66–68

Overall, CpG is a promis-

ing therapeutic agent with many potential applications.

However, additional research is required to completely com-

prehend its mechanism of action and determine its human

safety and eﬃcacy.

3. Classiﬁcation of spherical

particles/nanoparticles for potential

complexation with nucleic acids

The development of NPs provides diverse platforms for deliver-

ing drugs with enhanced eﬀects. However, it is reported that

NP morphology (viz., shape and size) can directly aﬀect their

response in biological systems.

Amidst them, spherical NPs

such as metallic NPs, polymeric NPs, protein, nucleic acids,

and lipid-based complexes having a shape in common are the

most promising drug delivery agents and therapeutic probes.

It has been reported that under in vivo conditions, the NPs

behave diﬀerently from biomolecules in therapeutic appli-

cations, particularly in vaccine delivery.

(1) The larger surface

area to volume ratio and permeability of these nanocomplexes

allow eﬃcient and targeted delivery of the cargo (DNA/RNA

etc.) and reduce unwanted side eﬀects.

(2) Nanocarriers can

be tailored to slowly release loaded their cargo, providing con-

trolled and sustained release within the targeted tissue or

organ. (3) In biological systems, small molecules face chal-

lenges such as degradation by enzymes and other biological

processes. Nanostructures can encapsulate the therapeutic

molecules, protecting them from degradation and ensuring

their eﬀective and targeted delivery. This is especially impor-

tant for fragile molecules like DNA and RNA used in nucleic

acid vaccines.

(4) Naked DNA or RNA can sometimes trigger

unwanted immune responses. Nanocarriers can shield the

nucleic acids, overcoming such reactions and promoting a

more focused immune response toward the target antigen.

73,74

Overall, under in vivo conditions, nanostructures provide dis-

tinct advantages over small molecules. Their ability to deliver

drugs directly to target sites, oﬀer controlled release, and

protect sensitive molecules makes them a powerful tool for

improved treatment eﬃcacy and reduced side eﬀects.

Table 2 shows the merits and demerits of these drug deliv-

ery vehicles. One can choose the ideal carrier to deliver par-

ticular drugs/genes based on these advantages and disadvan-

tages. A perfect carrier transports the drug to its target site and

releases it at the site of action. Several conditions must be

addressed, including specific and selective interactions,

suﬃcient drug delivery, and sustained drug release at the tar-

geted area. For example, nanoparticles can be made from

various materials to encapsulate hydrophobic and hydrophilic

drugs with increased stability. Similarly, polymeric NPs can be

used to encapsulate hydrophobic and hydrophilic drugs and

can be designed to release drugs in a controlled manner over

time. Lipid-based drug delivery agents can encapsulate hydro-

phobic drugs, protecting them from degradation and helping

them reach the targeted area in the body.

Review RSC Pharmaceutics

Table 2 Merits and demerits of advanced delivery systems

no Delivery agent Advantages Disadvantages Ref.

1 Inorganic NPs ✓Ease of synthesis and surface functionalization •Intracellular toxicity 75

✓Enhanced intracellular uptake •Rapid elimination by the

reticuloendothelial system

✓Able to entrap both hydrophilic and hydrophobic drugs •Lack of clinical trials

✓Enabled active and passive targeting •Poor biodegradability

✓Photoluminescence properties •May trigger the immune system

✓Stability (w.r.t. wide range of pH and temperatures)

Nobel metal (AuNPs,

AgNPs) NPs

✓Strong biocompatibility •Expensive for large-scale applications 76

✓Tunable optical properties •Potential toxicity concern

✓High surface-to-volume ratio •Stability issues due to aggregation at

elevated temperatures✓High binding aﬃnity

Quantum dots (QDs) ✓High quantum yield •High cytotoxicity 77

✓Resistance to photobleaching as compared with

traditional organic dyes

•Exponential decrease in fluorescence and

blinking of diﬀerent QDs

✓Size-dependent emission wavelength from visible to NIR

region

•Instability and elevated hydrodynamic

diameter on interaction with serum

proteins

Silicon NPs (SiNPs) ✓Good biocompatibility and biodegradable •Low quantum yield 78

✓Tunable optical properties •Potential toxicity concerns

✓Ease of functionalization

✓Thermal stability

Carbon nanomaterials

(CMs)

✓Tunable photoluminescence •Toxicity concerns due to ROS generation,

inflammation, DNA damage 79

✓Photostability •Limited understanding of the long-term

eﬀect on human health

✓Ease of fabrication •Limited control over size and structure

✓Economical

✓Eco-friendly, and biocompatible

Iron oxide NPs ✓Biocompatibility •Toxicity concerns 80

✓Magnetic properties •Stability issues due to aggregation

✓High surface-to-volume ratio •Long-term eﬀects

✓Tunable properties •Costly production

2 Polymeric NPs ✓Biocompatible and biodegradable •Burst eﬀect 81–83

✓Able to entrap both hydrophilic and hydrophobic drugs •Limited drug loading

✓Ease of surface functionalization •Deep knowledge of polymer–receptor

molecular interactions required

✓Controlled and sustained release •Limited knowledge about long-term side

eﬀects✓Protect the drug from metabolic degradation

✓Prolonged residence time

3 Dendrimers ✓Increasing solubility of highly lipophilic drugs •Not a suitable candidate carrier for

hydrophilic drugs 84

✓Tunable physicochemical properties •Cellular toxicity

✓Ease of surface functionalization for targeted drug

delivery

•Elimination and metabolism depend on

the generation and materials

✓Covalently associating drugs •High cost for their synthesis

4 Nanofibers ✓Large surface area •Toxicity 85 and

86✓Biocompatibility •Degradation

✓Encapsulation and targeted delivery •Expensive for large-scale production

✓Tissue engineering

5 Proteins/peptides ✓Various anticancer eﬀects •Size influenced pharmacokinetics 83

✓High cell permeability •Short half-life

✓Low systemic toxicity

✓Improved target selectivity

✓Bypass biological barriers

6 SNAs ✓High specificity and potency •Low yield and high cost of synthesis 87 and

88✓High drug loading eﬃciency •Low stability w.r.t. temperature and UV

exposure

✓Biocompatibility and prolonged circulation time •Limited targeting ability

✓Dense packing and delivery of therapeutics •Limited knowledge about their behavior

in in vivo systems✓Resistant to nuclear degradation

✓Eﬀective gene regulation

7 Liposomes ✓Biocompatible and biodegradable •Poor stability 83

✓Able to entrap both hydrophilic and hydrophobic drugs •Short shelf life and instability in

circulation

✓Controlled release •A special storage system is needed

✓Protect the drug from metabolic degradation. Prolonged

circulation time

•Batch-to-batch variation in the size of

liposomes

✓Low systemic toxicity

RSC Pharmaceutics Review

3.1 Inorganic NPs

Inorganic NPs are vital in biomedical applications, viz., bio-

imaging, therapy, diagnosis, and drug delivery.

89,90

Inorganic

NPs are mainly metal oxides, semiconductors, and noble

metals with sizes ranging from 1–100 nm. These NPs pos-

sessed intrinsic physical, optical, magnetic, and electrical pro-

perties due to the characteristics of the core material.

Additionally, these properties can be controlled by tailoring

the size, shape, structure, and composition to achieve

enhanced sensing and therapeutic eﬀects.

NPs gain positive

responses as nanocarriers because of their tunable properties,

improved eﬃcacy, and decreased side eﬀects by boosting their

targeting ability.

92,93

The loading can be done via four

methods: physical adsorption, electrostatic interaction, encap-

sulation inside the NP core, or covalent binding. For example,

gold NPs (AuNPs) are well known for their therapeutic and

delivery capabilities due to their stability, inertness, high

binding aﬃnity to thiols, amines, and disulfides, and ease of

functionalization with ligands such as drugs, proteins, and

nucleic acids. Excellent fluorescence due to the surface

plasmon resonance eﬀect resulted in absorption in the visible

and NIR regions. Therefore, it is essential in diagnosis and

sensing applications.

Also, depending upon the shape and

size of the AuNPs, the free electrons on the surface of AuNPs

oscillate continually, granting them photothermal

properties.

95,96

Thiolate NAs can be easily conjugated to

AuNPs via covalent and electrostatic interactions between

sulfur and gold.

Gracezyk et al. (2021) developed an AuNP

carrier to deliver siRNA by functionalizing AuNPs with a thiol-

modified tectoRNAs trimer (structural RNA). They applied this

conjugate to regulate the CopGFP expression in MDA-MB-231

GFP/RFP cells (Fig. 1a).

The cellular uptake of AuNP: to

tectoRNA conjugate was determined by TEM studies, showing

Table 2 (Contd.)

no Delivery agent Advantages Disadvantages Ref.

8 Lipid-based

nanocomplexes (LNPs and

SLNs)

✓Production on a large-scale industry level •Limited drug loading for SLNs 84

✓Easy sterilization •Instability in the bloodstream

✓Able to entrap both hydrophilic and hydrophobic drugs •SLNs tend to gelation

✓Able to target specific cells or tissues •Polymorphic transition for SLNs

✓Modulated and controlled release •Particle size growth and drug expulsion

during storage✓Low toxicity due to their biocompatible and

biodegradable components and the absence of organic

solvents in their process

✓Protecting drugs from environmental conditions

✓Low cost compared with liposomes

Fig. 1 (a) Impact of AuNP:structural RNA complex on MDA-MB-231 GFP/RFP cells. In vitro studies show the regulation of CopGFP expression in the

(A–C) nanoparticle dimers and (D−F) “non-targeting”scrambled nanoparticle dimers. Sixteen HBE cells express only keratin 8, and MRC 5 cells

express only vimentin. A549 expresses both keratin 8 and vimentin. A ﬂuorescence signal corresponding to the presence of keratin 8 mRNA (A),

vimentin mRNA (B), and both vimentin and keratin 8 mRNA (C) are observed. When incubated with nanoparticle dimers designed with “non-target-

Review RSC Pharmaceutics

their presence in the cytoplasm both inside the cytosol and

membrane structure. Also, the prepared AuNP:tecto RNA con-

jugates eﬀectively regulated gene expression, as demonstrated

by GFP expression studies using fluorescence techniques.

Kyriazi et al. (2018) synthesized a DNA–AuNP dimer-based

multifunctional platform for mRNA sensing and targeted drug

delivery.

The synthesized DNA–AuNP dimer specifically

recognizes keratin 8 and vimentin mRNAs in keratin 8-expres-

sing 16HBE (epithelial cells), vimentin-expressing MRC 5

(mesenchymal cells), and A549, showing the expression of

both keratin 8 and vimentin respectively. Furthermore, two

anticancer drugs, doxorubicin (Dox), which detects keratin

8 mRNA, and mitoxantrone (MXT), which detects vimentin

mRNA, were intercalated into a DNA duplex, resulting in cell

death in response to specific mRNA signatures. Contrary to

this, the scrambled NPs, without having recognition sites for

targeted mRNA, failed to release the loaded drugs (Fig. 1b).

Another class of inorganic NPs includes semiconductor

quantum dots (QDs) due to their nanosize showing size-

tunable 3D quantum confinement eﬀects, thus giving rise to

exquisite optical properties.

99–101

QDs are typically made of

semiconducting materials such as group II–VI elements (CdS,

CdSe, CdTe, ZnS, ZnSe, ZnTe), group III–V elements (InP or

InAs), group I–III–VI

elements (CuInS

, AgInS

), group IV–VI

elements (PbS, PbSe, PbTe), or group IV elements (C, Si,

Ge).

102–105

QDs are important fluorophores as they provide

emission in the UV, visible, and near-infrared ranges with an

impressive quantum yield compared with traditional organic

dyes, with drawbacks including photobleaching and poor

signal intensity.

106,107

All these properties of QDs make them

suitable probes for optical bioimaging and targeted molecular

sensing. For instance, Ma et al. (2019) designed a CdTe:Zn

QD nanobeacon conjugated with black hole quencher (BHQ1)

and phosphorothioate co-modified DNA by hydrothermal syn-

thesis for single RNA detection and imaging (Fig. 2a(A)).

108

This nanobeacon was highly sensitive and eﬃciently detected

low-abundance nucleic acids in live cells via FRET. QDs func-

tionalized with BHQ1, and single DNA were applied to detect

and image single HIV-1 RNAs in live HIV-1 integrated cells

(Fig. 2a(B, C)). Similarly, gene silencing in tumor cells of the

central nervous system was done using siRNA-loaded polyethyl-

enimine (PEI) functionalized CdSSe/ZnS QD-based nano-

carriers.

109

siRNA-loaded PEI-CdSSe/ZnS QDs eﬃciently target

human telomerase reverse transcriptase (TERT). Two glioblas-

toma cell lines, U87 and U251, after transfection showed a

decrease in gene and protein expression levels of TERT with a

high level of gene transfection eﬃciency within 48 h.

The rapid development of silicon nanostructures provides a

potential class of sensitive sensors and therapeutic agents for

real-time diagnosis and therapeutic applications.

110–112

Silicon

NPs (SiNPs) are indirect band gap semiconductors and thus

exhibit longer excited-state lifetimes; after entering the cellular

system, SiNPs are biodegraded into silicic acid (nontoxic com-

pound) and easily excreted out of the body without showing

any sign of toxicity.

78,113

Chaix et al. (2019) reported amine-

functionalized porous SiNPs for the loading and improved

delivery of pDNA

114

(Fig. 2b). In vitro studies suggested that

the SiNPs showed better biocompatibility and successful trans-

fection of up to 10

RLU mg

−1

proteins in HEK 293 cells.

Carbon nanomaterials (CNMs), also called green NMs, are

admirable fluorescent NMs with fascinating characteristics

such as tunable photoluminescence, photostability, ease of

fabrication, economical production, eco-friendliness, and bio-

compatibility.

115

In contrast to QDs, the mechanism behind

the fluorescence property of CNMs involves π-plasmon and

surface defects generated from radiative recombination of the

surface-confined electrons and holes.

116

Wang et al. (2014) syn-

thesized fluorescent carbon dots (CDs) for simultaneous

imaging and eﬃcient siRNA delivery for cancer therapy.

117

PEI-functionalized CDs were applied for the adsorption of sur-

vivin siRNA. After transfection for two hours with siRNA-

loaded CDs/PEI complexes, MGC-803 gastric cancer cells

showed blue fluorescence in the cytoplasm, thus suggesting

the internalization of loaded siRNA into the cancerous cells

(Fig. 2c). Furthermore, the expression of survivin mRNA in

MGC-803 cells was downregulated to 96.4 ± 8.7 when exposed

to siRNA-loaded CD/PEI complexes. Iron oxide NPs (IONPs),

with their remarkable superparamagnetic properties and excel-

lent surface-to-volume ratio, have attracted worldwide inter-

est.

Magnetic NPs are widely used for magnetic particle

imaging, magnetic resonance imaging, hyperthermia, cell

tracking, targeted genes, and drug delivery.

118,119

Iron oxide

NPs are comparatively less toxic and cytocompatible, thus

allowing clinical translation. IONPs are usually synthesized

from iron oxides such as maghemite (γ-Fe

) and magnetite

(Fe

), metal alloys (e.g., FeCo and FePt), as well as the

doping of magnetically susceptible elements (e.g., MnFe

and CoFe

120,121

Eﬃcient gene (pDNA) delivery to

mesenchymal stem cells was accomplished using positively

charged, PEI-coated IONPs.

122

The uniform and narrow-sized

(15 nm) IONPs exhibit good magnetic properties. In vitro

studies suggested that IONPs under the external magnetic

field eﬃciently delivered 99% of the loaded pDNA within

30 min, followed by nuclear importing of the carried genes,

resulting in enhanced gene expression of the treated cells. The

caveolin-mediated pathway facilitated the internalization of

pDNA-loaded IONPs into the cytoplasm. Zhang et al. (2010)

designed PEI-coated IONPs and applied them to deliver inter-

fering RNA (siRNA) GFP plasmid via an external magnetic

force in 3D cell culture (NIH 3T3 cells).

123

The NPs facilitated

64% and 77% transfection eﬃciency for siRNA and GFP

plasmid, respectively. These transfection complexes signifi-

cantly reduced the GFP-expressed cells’growth, with 80–82%

silencing eﬃciency. Furthermore, these complexes delivered

four toxic shRNA to the 3D cell culture, enhancing cell death

(41–51%) (Fig. 2d).

Other commonly used inorganic NPs include silica NPs, which

are hydrophilic and porous, have access to surface functionali-

zation due to silane groups, and are widely used for drug delivery,

biomolecule conjugation, and many more applications.

However, for in vivo applications, the toxicity of these in-

organic NPs is an inevitable issue among researchers.

RSC Pharmaceutics Review

Additionally, the cellular uptake, circulation, and clearance of

NPs depend upon the physicochemical properties of the NPs.

After entering the bloodstream, NPs come across the biological

barrier composed of lipids, proteins, and other components.

The interaction of NPs with biomolecules resulted in the for-

mation of biomolecule coronas around the NPs before reach-

ing the target site, thus influencing the biological fate of the

NPs.

100

To circumvent these issues, researchers developed bio-

compatible surface modification approaches to inorganic NPs

to avoid or decrease the nonspecific interactions of these NPs

with biomolecules and increase prolonged accumulation at

the site of interest.

3.2 Polymeric NPs

Polymeric nanoparticles have shown great potential in recent

years due to their small size, ranging from 1–1000 nm.

Polymeric nanoparticles are mostly spherical with a solid

structure. The main advantages of polymeric NPs (PNPs)

Fig. 2 (a) Schematic illustration of QD-NBs for HIV-1 genomic RNA detection. (A) Schematic diagram of the QD-NBs’preparation; schematic illus-

tration of QD-NBs for (B) HIV-1 genomic RNA detection in living cells, and (C) ﬂuorescence labeling of single virus particles. Adapted with per-

scanning microscopic images of MGC-803 cells incubated with Cdots@PEI or Cy3-siRNA for 2 h, or Cy3-siRNA-Cdots@PEI complexes for 2 and 5 h.

Review RSC Pharmaceutics

include (1) the high molecular weight and polyvalent nature of

these molecules, which enable the encapsulation and delivery

of bulky and long-chain NAs,

124

(2) biocompatibility and bio-

degradability: PNPs easily break down inside the body into

water and carbohydrates and hence can be quickly eliminated

from the body,

125

(3) facilitation of successful loading and con-

trolled drug delivery,

126,127

(4) retention of the bioactivity of

drugs or biomolecules by evading the immune system, thus

enhancing their bioavailability and therapeutic potential, and

(5) the provision of a platform for ligand functionalization,

resulting in the targeted and stealthy delivery of drugs.

Polymeric nanoparticles can be synthesized by natural and

synthetic polymers.

128

Natural polymers, namely chitosan, hya-

luronic acid, starch, alginate, cellulose, and lignin, and syn-

thetic polymers, viz., polylactide-co-glycolide (PLGA), polylac-

tides (PLA), polyethylenimine (PEI), polyethylene glycol (PEG),

polycaprolactones, and polyacrylates, have been widely

explored for the synthesis of PNPs.

125,126,128

The most

common polymeric NPs include nanocapsules (polymeric cap-

sules surrounding a cavity) and nanospheres (solid matrix).

For instance, a nanovehicle based on cationic cyclodextrin-

polyethyleneimine 2k conjugate delivered mRNA encoding HIV

glycoprotein120 for treating HIV-1.

129

The delivery system

enhanced the intranasal delivery of mRNA by crossing the

nasal epithelial barrier through intracellular pathways, thus

resulting in a solid anti-HIV immune response. Biodegradable

chitosan-alginate 3D porous injectable gel was designed for

in vivo mRNA vaccine delivery.

130

Increased levels of IFN-α

secretion, luciferase reporter protein expression, and T-cell

proliferation were observed from mRNA lipoplex-loaded gel

scaﬀolds compared with the systemic injection of naked

mRNA and mRNA:lipoplex.

A nanovehicle based on PLGA-encapsulated antisense

microRNA-21 (miRNA-21), known to be overexpressed in glio-

blastoma cells, was applied for the improved therapeutic eﬀect

of temozolomide (TMZ) on glioblastoma cells (Fig. 3a).

131

Enhanced drug delivery and sustained gene silencing of

miRNA 21 were observed in glioblastoma cells, namely U87

MG, LN229, and T98G cells. This nanovehicle also showed a

significant decrease in cell viability (p< 0.001) with a 1.6-fold

increase in cell arrest at the G2/M phase in the TMZ-treated

cells (Fig. 3b–d). Furthermore, the intracellular co-delivery of

the nanocomplex and TMZ in glioblastoma cells resulted in a

67% and 15% enhancement in the expression of miRNA-21

targeted phosphatase and tension homologue (PTEN) genes

and apoptosis-associated caspase-3 respectively (Fig. 3e). The

PLGA–SNA complex was designed to accommodate the chemo-

therapeutic drug coumarin spatially and nucleic acid to inde-

pendently improve the controlled loading and tunable release

of encapsulated moieties.

132

In vitro studies performed on

RAW blue cells to confirm the immunotherapeutic response of

PLGA–SNAs depicted dose- and time-dependent activation of

TLR9. PLGA–SNAs were cytocompatible at concentrations

ranging from 10 × 10

−9

Mto2×10

−6

M. Also, the cellular

uptake eﬃciency of PLGA–SNAs was tenfold higher than their

linear counterpart and at a shorter time of 0.5 h. However,

PNPs also suﬀer from drawbacks, including toxicity and a high

risk of particle aggregation. The FDA approves very few PNP-

based drugs for clinical applications.

133

Dendrimers or “dense stars”are globular, hyperbranched

3D structures, and their physicochemical properties can be

controlled for the desired application. Dendrimers can be syn-

thesized by divergent or convergent techniques.

134,135

The

Fig. 3 (a) Phase contrast microscopic images of control and antisense

miR-21 transfected cells treated with 500 μM TMZ. In vitro cell viability

MTT assays on control and antisense miR-21 transfected (b) U87 MG

cells, (c) LN 229 cells, and (d) T98G cells treated with diﬀerent concen-

trations of TMZ. (e) Cellular pathway analysis of antisense miR-21 and

TMZ co-treatment on U87 MG cells. Adapted with permission from ref.

tation of PAMAM dendrimer/pDNA-p53 nanocarrier preparation for

cancer gene therapy. Adapted with permission from ref. 139. Copyright

2021, American Chemical Society.

RSC Pharmaceutics Review

remarkable properties of dendrimers make them suitable

vehicles for the delivery of nucleic acids. For instance, the poly-

cation nature of dendrimers provides multiple sites for the

electrostatic binding of the negatively charged phosphate back-

bone of NAs, resulting in solid DNA complexation. Also, the

formation of the dendrimer–NA complex protects against

nuclease degradation of the attached NAs. It is speculated that

the presence of tertiary amines in the structure of dendrimers

enhanced the cellular uptake and delivery of NAs through

endosome escape via the sponge eﬀect.

136

Other merits

include the attachment of biomolecules or ligands to the func-

tional groups available on the exterior surface. In contrast,

drugs and small cargo can be encapsulated in the interior of

the dendrimer.

133

Various dendrimers are studied for the

delivery of NAs, such as poly L-lysine, triazine, polyglycerol,

poly(propyleneimine), and poly(amidoamine) (PAMAM) based

dendrimers. At the same time, PAMAM is the most investi-

gated nanocarrier because of facile synthesis and functionali-

zation.

137

Palombarini and coworkers (2021) demonstrated the

targeted delivery of miRNA to myeloid leukemia cells (which

are otherwise challenging to transfect) by incorporating

nucleic acid with ferritin-poly(amidoamine) (PAMAM) dendri-

mer NPs.

138

The cellular internalization of this nanocomplex

drives morphological changes and enhances the expression of

the retinoic acid receptor, alpha (RARα), an early hallmark of

granulocytic diﬀerentiation. Mekuria et al. (2021) developed a

nanocarrier by covalently binding PAMAM dendrimer with

4,4′-dithiodibutryic acid (DA) to successfully deliver p53-pDNA.

The nanocarrier reflected a 2.3- and 2.1-fold increased gene

transfection in 4T1 and mouse breast cancer cells.

139

Furthermore, in vitro and in vivo studies showed an upregu-

lated expression of mRNA and p53 and p21 protein and down-

regulation of cyclin-D1 and CDK-4 protein, thus facilitating

cell cycle arrest in the G1 phase (Fig. 3f).

Dendrimers are ideal for delivering NAs with enhanced

stability and improved cellular internalization compared with

other therapeutics. However, regarding the safety concerns,

the toxicity of these dendrimers is still unaddressed. The

strong cationic amine groups on the surface of dendrimers

resulted in solid binding with the negatively charged cell mem-

brane, which led to destabilization, disruption of cell com-

ponents, and eventually lysis.

140

Nanofibers are a promising class of biomaterials for nucleic

acid delivery, oﬀering several advantages including a large

surface area, biocompatibility, encapsulation and targeted

delivery (gene, growth factors, protein, and peptide delivery),

and scaﬀolding for tissue engineering.

Nanofibers can be

synthesized by diﬀerent methods, namely, phase separation,

electrospinning, and physical and chemical fabrication.

Various natural, semisynthetic, and synthetic polymers are

widely used for their synthesis. Furono et al. (2022) reported

plasmid DNA delivery using horseradish peroxidase cross-

linked gelatin nanofibers.

141

Nanofibers immobilized with

Lipofectamine/pDNA resulted in the transfection of pDNA

delivery in HEK293 cells. Furthermore, genome-editing mole-

cules including Cas9 protein and guide RNA (gRNA) were

expressed in nanofiber-treated HEK293 cells, resulting in gene

knock-in and knock-out. Polycaprolactone nanofiber-encapsu-

lated siRNA showed controlled release for up to 28 days with

successful transfection of the treated HEK 293 cells.

142

The

cells showed enhanced cellular uptake and GAPDH gene silen-

cing of 61–81%. Nanofibrous scaﬀolds made up of collagen

type 1 were used for the controlled and long-term delivery (at

least 5 months) of siRNA/silica NPs.

143

In vivo studies revealed

that the nanofiber-based scaﬀolds showed more eﬀective gene

silencing (p< 0.05) as compared with traditional bolus deliv-

ery. An in vivo biodistribution study revealed that siRNA stayed

confined up to ∼290 μm from the implants. As compared with

negative scrambled siRNA therapy, a reduction in fibrous cap-

sules of ∼45.8% was observed after 4 weeks.

Along with the advantages, nanofibers do have some draw-

backs that researchers are working to overcome.

(1)

Nanofibers may have inherent toxicity or cause inflammatory

responses in the body. (2) Depending on the material, nano-

fibers may degrade too quickly or too slowly, aﬀecting the

release profile of the drugs they carry. (3) Manufacturing nano-

fibers for large-scale clinical use can be complex and

expensive.

3.3 Proteins/peptides

Proteins, a class of natural biomolecules, stand out as an

attractive biocompatible substitute for synthetic polymers in

nanomedicine due to their biodegradability, natural abun-

dance, mild synthesis, and fast metabolization.

144,145

Additionally, proteins showed remarkable chemical modifi-

cation properties due to their surface abundance of functional

groups such as carboxyl, amine, and hydroxyl. The amphiphi-

lic nature makes them amenable for small molecules, metallic

NPs, and hydrophobic and hydrophilic drugs.

145,146

Protein-

based NPs, when administered inside the body, showed weak

immunogenicity, were eﬃciently degraded by the enzymes and

were eliminated through hepatic clearance. Abraxane is an

FDA-approved drug composed of paclitaxel-bound albumin-

based NPs used to treat metastatic breast cancer by inhibiting

the mitosis of cancer cells.

147,148

The drug avoids hypersensi-

tivity due to albumin protein, a major drawback of traditional

anticancer drugs. Protein NPs can be easily synthesized via

electrospray, emulsion, and desolvation processes using

natural proteins, namely albumin, fibroins, 30Kc19, gelatin,

lipoprotein, legumin, zein, gliadin, and ferritin proteins.

144,149

Peptides are short chains of amino acids linked by a

covalent amide bond (peptide bond). They exhibit remarkable

sequence and functional diversity, merits employed to design

spherical NPs via a self-assembly approach.

150

Several peptides

are currently used for biomarker imaging, targeted drug and

gene delivery, bioprinting, wound healing, and tissue

engineering.

151,152

Diﬀerent peptides such as nuclear localiz-

ation, tumor-targeted, and cell penetration peptide sequences

are extensively reported for biomedical applications. Jia et al.

(2020) reported enhanced siRNA delivery and improved gene

silencing using hyaluronic acid (HA)-modified transmembrane

peptide octa-arginine (R8)-based R8-bipolar (HA-bibola/siRNA)

Review RSC Pharmaceutics

and R8-monobola (HA-bola/siRNA) amphiphilic nanocom-

plexes.

153

Cell viability studies reflected better cytocompatibil-

ity of HA-bibola/siRNA compared with HA-bola/siRNA and

control samples (PEI/siRNA) in 4T1 cells. Additionally, HA-

bibola/siRNA showed enhanced cell uptake eﬃciency and

down-regulation of Bcl-2 protein expression due to the pres-

ence of cell-penetrating peptides on the surface of the nano-

complex. In vivo, studies reflected higher antitumor eﬃcacy,

improved targeted ability, and increased Bcl-2 gene suppres-

sion in 4T1 tumor-bearing Balb/c mice.

3.4 Spherical nucleic acids (SNAs)

The primary function of nucleic acids includes storing and

transduction of genetic information. Nucleic acid-based (DNA,

RNA, CRISPR/Cas9 gene editing system) spherical NPs hold

great significance in the biosensing, therapy, and silencing of

various diseases ranging from viral infections to neurological

disorders, cardiovascular diseases, and cancer by taking

advantage of cellular pathways.

154–156

In the present era, thera-

peutic technologies related to nucleic acids are at the forefront

of fighting the COVID-19 pandemic around the globe.

157,158

They consist of highly compact self-assembled oligonucleotide

layers oriented in 3D geometries based on typical Watson–

Crick base-pairing.

119,120

Selective and precise base-pairing of

DNA/RNA NPs diﬀerentiates them from traditional bio-

molecules, which might be otherwise diﬃcult to design with

such ease.

159

Various chemical modifications (nucleotide

sequence, sugar, or phosphate backbone modification), bio-

compatibility, and programmable therapeutic approaches are

other fascinating properties of these nanostructures.

160

Most

nucleic acid-based NPs interact via complementary base

pairing with their target molecules, thus resulting in specific

and rapid action.

161

Spherical nucleic acids (SNAs) comprise

(1) the outer shell of densely packed nucleic acid radially

encasing the contents, and (2) an inner core of NPs. They were

initially made of DNA shells and AuNP cores.

161,162

Since then,

various inorganic nanoparticles such as Ag,

163

QDs, magnetic

NPs,

164

silica,

165

organic nanocomposites such as polymers,

proteins, and liposomes,

166,167

and hybrid structures of in-

organic–organic materials were used to explore diﬀerent bio-

medical applications. The three-dimensional architecture of

SNAs provides them with unique physicochemical properties

and makes them superior to their linear counterparts.

The

spherical shape allows the dense packing of the oligo-

nucleotide into a limited space.

168

This compact structure of

SNAs leads to an enhanced electric charge, increasing the

stability of the SNAs and providing resistance to nuclease

degradation and prolonged cell accumulation via scavenger

receptor engagement and endocytosis.

169

SNAs can easily

evade the immune system’s attack due to strong electric

charges because of the dense packing. Also, the interaction of

SNAs with receptors on the cell surface resulted in easy pene-

tration through the cell, tissue membranes, and even the

blood–brain barrier.

170

Melamed et al. (2018) suggested that

the spherical architecture of SNAs functionalized with poly-

ethylenimine has improved siRNA-mediated GFP gene silen-

cing 10-fold.

171

Wang et al. (2019) reported an SNA-based

vaccine for treating a mouse tumor.

172

SNA co-delivered CpG

oligonucleotide (adjuvant) and peptide (antigen) to generate

an antitumor immune response. The results suggested that

the vaccine could increase the survival rate to 31 days, also

delaying tumor growth by 15 days. SNAs eﬃciently promote

gene regulation for the treatment of skin diseases.

173

Randeria

et al. (2015) showed the application of siRNA-encapsulated

SNAs in impaired wound healing by downregulating ganglio-

side GM3 synthase (GM3S) in diabetic mice.

174

The expression

of GM3S was reduced by >80% at the wound site via the siRNA

pathway, and the wound was observed to heal within 12 days

in the SNA-treated mice.

174

Although nucleic acid-based drugs are in the infant stages

of clinical trials, they have revolutionized therapeutics’fate in

recent years. Despite the remarkable merits of SNA-based

therapeutics, challenges still hinder their further clinical and

translational applications.

4. Classiﬁcation of lipid

nanocomplexes for eﬃcient delivery

of NAs

The clinical application of NPs depends on their successful

delivery to the disease site. The eﬀectiveness of synthesized

NPs in treating diseases depends upon their therapeutic

response, which can be achieved by delivering a required

dose to the targeted site. During their journey to a target site,

NPs must evade multiple biological barriers at diﬀerent sites,

including (1) phagocytosis by liver cells while NPs are circu-

lating in the blood.

175

(2) Even after reaching the target site,

NP entry into the disease site is restricted by the endothelial

cell walls. (3) The immune system also recognizes and

destroys foreign components (NPs) and vectors (spherical

DNA/RNA containing genetic information).

176

(4) The intern-

alization of NPs in the cell membrane or nucleus enhanced

the eﬀect of the former. The overall eﬀectofthesebarriers

restricted the penetration of most NPs into the targeted cell

or organ and thus reduced their therapeutic eﬃciency.

Recently, remarkable progress has been made in designing

and developing delivery systems that enhance the therapeutic

trajectory of drugs, inorganic NPs, and DNA/RNA-based

genetic drugs.

Lipid-based delivery cargos, namely micelles, liposomes,

and lipid nanoparticles (LNPs), are promising delivery agents

as they can protect nanomaterials from degradation and

enhance circulation by avoiding early clearance by the

immune system.

177,178

Lipids are widely used nonviral delivery

agents and are fascinating because of their easy synthesis,

characterization, greater payload, homogeneity, biodegradabil-

ity, and marginal toxicity profile. A broad range of lipid-based

NPs is utilized to deliver NAs, including cationic lipids, ioniz-

able lipids, zwitterionic lipids, LNPs, liposomes, and solid

lipid NPs (SLNs) (Table 3).

RSC Pharmaceutics Review

Lipid-based delivery agents, viz., micelles, liposomes, and

lipid NPs, have been extensively studied to encapsulate and

deliver NAs, primarily due to their ease of synthesis and inter-

action with NAs (Fig. 4a).

195

The essential components of FDA-

approved LNPs for NA delivery developed by pharmaceutical

companies include cationic or ionizable lipids, cholesterol, a

helper lipid, and a PEG-lipid (Fig. 4b and c), for example DLin-

MC3-DMA, PEG: PEG-2000-C-DMG (Alnylam), SM-102,

PEG-2000-DMG (Moderna), and ALC-0315(Pfizer/BioNTech)

ALC-0159 (Pfizer/BioNTech/Acuitas), along with DSPC and

cholesterol (Fig. 4d).

Moreover, lipid-based nanovehicles can be surface functio-

nalized to achieve the targeted delivery and release of thera-

peutics in a specific tissue or cell.

196,197

However, it is impor-

tant to consider the chemical properties of the lipids used in

NA delivery for eﬃcient drug development. One of the critical

parameters of lipids is their surface charge. Diﬀerently

charged lipids can have varying degrees of compatibility with

nucleic acids. In general, positively charged lipids tend to have

a higher degree of compatibility with negatively charged

nucleic acids, such as DNA and RNA, as electrostatic inter-

actions between the positive charges on the lipids and the

negative charges on the nucleic acids can help to stabilize the

interactions between the two. Conversely, negatively charged

lipids may have reduced compatibility with NAs, as the nega-

tive charges on the lipids may repel the negative charges on

the NAs. Also, it is well known that the charge of NPs alters

their tissue selectivity after intravenous administration. Taking

advantage of this approach, diﬀerent tissue-targeted LNPs

have been designed by incorporating cationic or anionic lipids

into the general composition of LNPs to tune their charge.

198

For example, using zwitterionic lipids, which have both a posi-

tive and negative charge, has been shown to enhance the stabi-

lity of NPs and improve in vivo eﬃcacy.

199

Additionally, using

lipids with a high melting point can help improve the NPs’

stability and enhance their ability to penetrate cell mem-

branes.

200

Another critical factor to consider is the size of the

NPs formed by the lipids. Smaller NPs have been shown to

penetrate cell membranes more eﬀectively and deliver NAs to

target cells.

201

However, it is also important to note that the

size of the NPs should not be so small that they are rapidly

cleared from the body by the immune system. Besides consid-

ering the lipids’structural and chemical properties, the NPs’

composition is also an essential consideration for NA deliv-

ery.

202

For example, using PEG as a surface coating on lipids

has been shown to improve the stability of the particles and

reduce their clearance from the body.

203

Additionally, using

other polymers, such as PEI or PLA, can also help improve the

stability and eﬃcacy of the lipid-based delivery agents. Overall,

the design of lipids for NA delivery is a complex process that

requires a thorough understanding of the structural, chemical,

and composition-based factors that influence their eﬃcacy.

Through the use of structure–activity relationships and careful

consideration of the properties of the lipids, it is possible to

design nanoparticles that can eﬀectively deliver NAs to target

cells and improve the eﬃcacy of gene therapy.

Table 3 A summary of lipid-based NA delivery systems and their biomedical applications

no Type of lipid vehicle Composition of vehicle Cargo Method of

synthesis Treatment Ref.

1 Cationic DOTAP/cholesterol mRNA, pDNA, and oligonucleotide Thin-film

evaporation Ovarian cancer 179

2 Cationic 9322-O16B/Chol/DOPE mRNA Chemical B-cell lymphoma 180

3 Cationic DOPE-stearylated octaarginine (STR-R8), DOTMA-YSK05,

cholesterol-GALA peptide pDNA Chemical Eﬃcient and selective delivery of pDNA to

the lungs 181

4 Ionizable cationic DLin-MC3-DMA/DLin-KC2-DMA/DODAP/DSPC/PEG-DSPE,

cholesterol siRNA Microfluidic pDNA transfection 182

5 Ionizable cationic C-12-200 (IL)/DOPE, cholesterol/PEG-lipid conjugate pDNA Microfluidic Cardiovascular diseases 183

6 Ionizable cationic DLin-MC3-DMA, DSPC, cholesterol, DMG-PEG2K mRNA Chemical Hepatic reticuloendothelial diseases 184

7 Ionizable DSPC, CHO, DMG-PEG

2000

siRNA Chemical Hyperlipidemia 185

8 Zwitterionic Phosphatidylcholine, DPPC, cholesterol pDNA Chemical pDNA transfection 186

9 Liposome DOTAP/DOPE/DSPE-PEG 6-Carboxyfluorescein-labeled 14-mer

oligonucleotide Ethanol dilution In vivo labeling of human microbiota 187

10 Cationic liposome DPPC/DOTAP/cholesterol GFP-mRNA Lipid film

hydration Neurodegenerative diseases 188

11 Protamine liposome DOTAP/cholesterol mRNA Chemical Colorectal cancer gene therapy 189

12 Lipid nanoparticles C-14-4 IL/DOPE/cholesterol/PEG mRNA Microfluidic Engineering of CAR T cells to kill cancer

cells 190

13 Lipid nanoparticles DLin-MC3-DMA/DAP/phospholipid/cholesterol/PEG Spherical DNA/RNA Ethanol dilution Organ-specific delivery of nucleic acid 191

14 Solid lipid NPs DOTAP/lecithin/cholesterol/lipopolysaccharide TNF-αsiRNA Ultrasonication Rheumatoid arthritis 192

15 Solid lipid NPs Stearic acid, soya lecithin pDNA, Dox Solvent

displacement Lung cancer therapy 193

16 Cationic solid lipid

NPs Peptide-cationic lipid CDO14 siRNA, upconversion NPs Thin-film

dispersion Bioimaging and gene therapy 194

Review RSC Pharmaceutics

4.1 Cationic lipids

Cationic lipids (CLs) are well characterized by their positively

charged hydrophilic head (such as quaternary ammonium,

amines), linker (such as ester, ether, disulfide), and negatively

charged hydrophobic tail (cholesterol, tocopherol). CLs are

well-explored for the delivery of negatively charged nucleic

acids. Cationic lipid, namely N-[1-(2,3-dioleyloxy) propyl]-N,N,

N-trimethylammonium chloride (DOTMA) bearing a quatern-

ary ammonium head group and unsaturated phosphatidyletha-

nolamine (DOPE), was first employed for nucleic acid delivery

for gene therapy by Felgner’s group in 1987.

204

The lipid

formed is still commercially available with Lepofectin and is

extensively used for the in vitro delivery of nucleic acids.

205

CLs

formed complexes with negatively charged phosphate groups

in the polynucleotides’DNA. The complexes are formed

through positively charged head groups via electrostatic inter-

actions, whereas hydrophobic tails exist as bilayers around

DNA.

206

This lipid envelope surrounding DNA provides resis-

tance against omnipresent nucleases and overcomes immuno-

Fig. 4 FDA-approved lipid-based structures contain some variation of the four essential components: cholesterol, a helper lipid, a PEG-lipid, and a

cationic or ionizable lipid. (a) Lipid-based structures can include micelles, which consist of a lipid monolayer, or liposomes, which consist of a

bilayer. Lipid nanoparticles comprise multiple lipid layers and lipid and nucleic acid microdomains. (b) and (c) In addition to the RNA payload, LNPs

often consist of cholesterol, a helper lipid, a PEG-lipid (all shown in part b), and a cationic or ionizable lipid (part c). (d) The molar ratios of the four

components constitute the FDA-approved Acuitas/BioNTech/Pﬁzer COVID-19 vaccine and patisiran, which delivers siRNA to the liver. Adapted with

RSC Pharmaceutics Review

genicity and mutagenicity. Additionally, it can easily combine

with other anionic sulfate groups of cell membrane proteogly-

cans, thus ensuring cell uptake. 1,2-Dioleoyl-3-(trimethyl-

ammonium) propane (DOTAP) and polyethylene-glycol-2000-

1,2-distearyl-3-sn-phosphatidylethanolamine (PEG-DSPE)

based cationic lipid was used to coat hydrophobic IONPs via

self-assembly synthesis.

207

The lipid-coated IONPs showed an

average size of 46 nm and improved delivery in three cell lines:

HeLa, PC-3, and Neuro-2a. Furthermore, in vivo, studies

revealed that the growth status of the tumor cells in Balb/c

mice was eﬃciently monitored through MR images, and the

magnetic property of IONPs was retained up to 15 days after

administration. Sun et al. (2022) demonstrated the eﬃcacy of

DOTAP/cholesterol-based CL NPs for successfully delivering

mRNA, pDNA, and oligonucleotide.

208

The nanocomplex was

stable for up to 60 days at 4 °C storage conditions without

aﬀecting transfection eﬃcacy. The eﬀective DOTAP/cholesterol

CL NP:mRNA ratio for in vitro studies was 62.5 µM lipid to 1 μg

mRNA. It was speculated that a lower lipid concentration

carried a lower surface charge, resulting in decreased cell inter-

action and endosome escape. In contrast, a higher concen-

tration might lead to cytotoxicity and inhibition of mRNA dis-

sociation from the nanocomplex.

Based on the chemical structure of a positively charged

head group, lipids can be classified into six classes, namely

quaternary ammoniums, amines (primary to quaternary),

amino acids or peptides, guanidiniums, heterocyclic head-

groups, and some uncommon headgroups.

177,209

Headgroups

containing charge and dimensions impact cell interaction,

endosome escape, and access to the target cell.

210

Linkers typically comprise non-biodegradable (e.g., ethers

and carbamates) and widely used biodegradable (e.g., esters,

amides, and thiols) functional moieties. Linkers form a junc-

tion between the headgroup and tails.

211

The linker aﬀects the

stability, biodegradability, cytotoxicity, and transfection

eﬃciency of LNPs.

The negatively charged, lipophilic aliphatic chains of

sterols form the last part of cationic lipids. The hydrophobic

tails elicit NP formation and potency by maintaining fluidity,

hydrophobicity, and fusion with the cell membrane.

209

Nevertheless, despite the advantages CLs oﬀer for the deliv-

ery of therapeutic cargo, researchers are motivated to continue

their development and optimization in preclinical research.

Their drawbacks include reduced in vivo eﬃcacy, low circula-

tion time, rapid elimination by RES, oﬀ-target accumulation in

the negatively charged cellular system, and unacceptable tox-

icity related to inflammatory responses, which has challenged

their clinical acceptance.

212–214

4.2 Ionizable lipids

Ionizable lipids (ILs) or ionizable cationic lipids are small

amphiphilic vesicles containing an ionizable protonatable ter-

tiary-amino head group, a spacer/linker, and a hydrophobic

moiety.

215

ILs showed the unique feature of electrostatic

charge dependency on the lipid pK

and pH of their environ-

mental surroundings and, thus, become positively charged at

acidic pH, ensuring the binding of therapeutic cargos while

maintaining a neutral charge at physiological pH, reducing

toxicity eﬀects.

216,217

ILs with a suitable pK

can modify their

electrostatic charge to ensure prolonged circulation and cyto-

solic release of therapeutics, elevated transfection eﬃciency,

access to targeted sites, and reduced toxicity. The transfection

eﬃciency of ILs depends mainly on the pK

, with an optimal

value of 6.2–6.5.

176,218

Ionizable lipids can be categorized into various subclasses

based on their structure.

(1) Unsaturated ILs: the degree of tail unsaturation sig-

nificantly impacts the fluidity and delivery eﬃciency of ILs.

The high unsaturation of lipid tails leads to nonbilayer lipid

formation, enhancing membrane disruption and cargo

release. Some common unsaturated ILs include DLin-MC3-

DMA (MC3), A18-iso5-2DC18, OF02, etc.

(2) Polymeric ILs: the structural basis of polymeric ILs is

the substitution of free amines on cationic polymers with alkyl

tails, and their hydrophobic aggregation results in improved

particle formation. Common examples include (1,2 bis(triclo-

san-10,12-diynoyl)-snglycero-3-phosphocholine) (DC8,9PC),

7C1, G0-C14 etc.

(3) Biodegradable ILs: the main objective of using

diﬀerent materials for biomedical applications includes

improved biocompatibility and biodegradability with reduced

toxicity and inappropriate accumulation inside the body.

202

Adding biocleavable ester bonds (simple structure, chemical

stability) to ILs provides biocompatibility via undergoing enzy-

matic hydrolysis in vivo. Maier et al. (2013) synthesized a

library of biodegradable ILs by incorporating ester bonds at

diﬀerent lipid positions.

202

Recently, anti-SARS-CoV-2 vaccines have also used ILs for

their formation.

219

The vaccines mainly comprise mRNA

motifs and LNPs composed of pH-sensitive ILs. Kim et al.

(2021) reported on the target delivery of RNA using engineered

ILs into specific liver cells, namely hepatocytes and liver sinu-

soidal endothelial cells.

220

The delivery of ILs to the targeted

site was ensured by controlling the size and PEG:lipid ratio

(Fig. 5a). Moreover, active targeting was achieved by adding

mannose to the ILs. In vivo, gene silencing studies showed the

selectivity of the engineered ILs towards the targeted liver cells

(Fig. 5b).

ILs are widely explored by modifying their domains, includ-

ing hydrophobic tails, hydrophilic heads, or linkers. Walsh

et al. (2013) reported a new class of IL with a lysine head group

linked to long-chain dialkylamine via an amide linker for

siRNA delivery.

221

The resulting pH-dependent ILs contain a

carboxylate group and two ionizable amines. This ionizable

lipid exhibits electrostatic charge-dependent membrane dis-

ruption advantages, successful in vitro siRNA transfection, and

enhanced siRNA-mediated knockdown in transfected HeLa

cells. Nucleic acid delivery was also accomplished using

diﬀerent aminoglycoside-derived ILs.

222,223

Lipid-based delivery systems are widely used for improved

chemotherapy for cancer treatment. Broma et al. (2019)

reported ILs as a Trojan horse in delivering AuNPs with a size

Review RSC Pharmaceutics

range of 5 nm for enhanced outcomes in the radiation therapy

of triple-negative MDA-MB-231 cells.

224

The ILs with the com-

position DLin-MC3-DMA/DSPC/cholesterol/PEG were used to

coat AuNPs. The complex of IL–AuNPs showed a ∼73-fold

increase in the uptake of small-sized AuNPs in cancerous cells.

4.3 Zwitterionic lipids

The zwitterionic lipids (ZILs) contain an equal number of co-

valently bonded anionic and cationic moieties.

225

ZILs have

gained the utmost attention in biomedical applications,

including drug delivery and increased uptake of loaded mole-

cules by reducing the adsorption of proteins on nanocarriers

in serum.

226

Obata et al. (2010) designed endosomal-pH-

responsive liposomes functionalized with glutamic acid-based

zwitterionic lipids for enhanced drug delivery applications.

227

The liposomes showed a positive zeta potential at lower pH

and became negatively charged at basic pH due to the carboxyl

group moiety in the glutamic acid. Furthermore, synthesized

pH-responsive lipids reflected high fusogenic potential with

the anionic membrane of cancer cells. Thus, it ensures the

Fig. 5 (a) Preparation of LNPs derived from ionizable lipids. (b) In vivo evaluation of ILs showed potent luciferase expression. Ex vivo organ images

showed that LNPs were mostly taken up into the liver. Liver histology image and transfection eﬃciency showed signiﬁcant tdTomato ﬂuorescence in

Synthesis and in vivo application of CP liposomes. (c) Tumor therapy eﬃcacy of CP liposomes (DDCP-c, DMCP-c, DPCP-c, and folate-CP) with

RSC Pharmaceutics Review

improved release of encapsulated Dox in HeLa cells and high

antitumor activity in vivo against a xenograft breast cancer

tumor. Cancer treatment with targeted and enhanced drug

delivery was ensured using a novel biorthogonal zwitterionic

lipid (choline phosphate (CP)) based liposome functionalized

with folic acid using a click reaction.

228

Furthermore, com-

pared with phosphatidylcholine (PC) based liposomes, supra-

molecular ionic pair interactions of zwitterionic lipid exhibit

adhesive characteristics with the cell membrane of cancer

cells, enhanced biocompatibility in normal cells, significantly

enhanced cytotoxicity and inhibition of tumor growth (Fig. 5c).

4.4 Liposomes

Liposomes are small spherical-shaped artificial vesicles syn-

thesized from cholesterol and nontoxic phospholipids.

Liposomes are widely explored as delivery agents due to their

size and ability to load both hydrophilic and hydrophobic

molecules.

229

Doxil was the first liposomal formulation

approved in 1995 by the FDA of the USA for the treatment of

refractory acquired immune deficiency syndrome (AIDS)-

related to Kaposi’s sarcoma.

230

The remarkable journey of lipo-

somes as delivery agents includes ligand-targeted delivery,

nucleic acid/gene delivery, and delivery of active drugs, poly-

mers, anesthetics, and antimicrobial agents.

231

Spherical lipo-

somes are readily taken up by irregular and distorted tumor

cells via an enhanced permeability and retention eﬀect, result-

ing in elevated drug distribution at the tumor site.

232

Curcumin and metformin-loaded DSPE-PEG2000-hyaluronic

acid liposomes were designed to target hepatocellular tumors

and drug resistance.

233

In vitro and in vivo studies revealed that

this formulation exhibits more potent antiproliferation and

antimetastasis. The inhibition of drug resistance and tumor

growth was attributed to the down-regulation of multidrug re-

sistance-related P-glycoprotein and the inducing epithelial–

mesenchymal transformation of tumor cells. Michel et al.

(2017) developed cationic liposomes to load mRNA and

improve cell transfection to treat alpha-1-antitrypsin

deficiency.

234

Liposomes showed a prolonged transfection

eﬀect with negligible cytotoxicity in A549 cells. Liposomes had

a long-acting transfection eﬀect on cells, resulting in increased

expression of a functional alpha-1-antitrypsin protein.

Dhaliwal et al. (2020) developed a cationic liposome-based

nanovehicle for intranasal delivery and potent mRNA transfec-

tion to a murine model’s brain.

188

The incubation of mRNA-

loaded cationic liposomes with J774.1 macrophage cells

showed stable GFP expression in the cytosol up to 24 h (Fig. 6a

and b). Furthermore, intranasal administration of mRNA-

loaded cationic liposomes in mice compared with control

Fig. 6 (a) GFP-mRNA transfection studies in J774A.1 macrophages using cationic liposomes at diﬀerent concentrations. (b) Quantiﬁcation of %

GFP-expressing cells. Lipofect_5 represents Lipofectamine (with 5 μg GFP-mRNA)-treated cells as a positive control, and the 0 μg GFP-mRNA/Lipo

group represents empty liposome-treated cells as a negative control (showed no GFP signal). (c) GFP-mRNA expression in mouse brain, 24 h post

Society.

Review RSC Pharmaceutics

(GFP-mRNA) and vehicle (liposome)-treated groups showed sig-

nificantly higher GFP expression by ∼15% (p< 0.05) up to 24 h

(Fig. 6c).

Lei et al. (2020) demonstrated colorectal cancer gene

therapy using a protamine/liposome-based delivery carrier

loaded with IL-15 mRNA (CLPP-mIL-5 complex).

189

Cytokine

IL-15 can be a cancer gene therapy due to its immune stimu-

lation characteristics. In vitro studies showed the successful

delivery of mRNA in C26 cells. The accumulation of mRNA in

cancer cells induced cytotoxicity and lymphocyte stimulation.

Following systemic delivery in three C26 murine colon cancer-

bearing mice, CLPP-mIL-5 complex inhibits cancer rates up to

70%, 55%, and 69% in abdominal cavity metastasis tumor,

subcutaneous, and pulmonary metastasis models, respectively.

4.5 Lipid nanoparticles (LNPs)

Lipid NPs are the highly advanced clinically viable vector for

NA delivery with Onpattro (patisiran) approval for amyloidosis

treatment and their application in COVID-19 vaccine around

the globe.

177,235

Lipid NPs are composed of four diﬀerent

lipids, namely (1) ionizable cationic lipid, (2) three neutral

helper lipids, namely phospholipid, (3) cholesterol, and (4)

PEGylated lipid.

236

In general, ionizable cationic lipids form

an electrostatic complex with anionic nucleic acid, enabling

intracellular uptake and endosomal escape. The stability and

fluidity of the complex are maintained by cholesterol.

Furthermore, phospholipids enhance the structural integrity

of the complex. PEGylated lipid is an important module that

maintains stability, enables cellular uptake, and protects the

lipid–NA complex from being captured by the protein

corona.

237,238

Billingsley et al. (2022) developed a sequential

library of biocompatible lipid NPs via microfluidic mixing to

successfully deliver mRNA to T cells (Fig. 7a and b).

190

Their

study revealed that lipid NPs composed of C14-4 ionizable

lipid improved the delivery of mRNA by a 3-fold increase in

primary human T cells. Additionally, lipid NPs with minimal

cytotoxicity induced chimeric antigen receptor (CAR)

expression and eﬀectively killed cancer cells (Fig. 7c). Ly et al.

(2022) developed lipid NPs using FDA-approved ionizable

lipids, namely, MC3, ALC-0315, and SM-102, and applied them

for the loading and delivery of self-amplifying mRNA (saRNA),

protein expression, and cytokine activation in vitro (triggering

diﬀerent levels of IL-6 response).

236

Furthermore, it was found

that PEG facilitates the encapsulation and stability of saRNA,

which is otherwise complicated to preserve as compared with

mRNA. The type of ionizable lipid highly influenced the

protein expression. Protein expression was highest in

ALC-0315, followed by SM-102, whereas MC3 failed to show

potent protein expression. The study provides an insight into

lipid NPs required for specific applications, including the

delivery of heavy RNA molecules (saRNA, Cas9), therapies

related to protein replacement, and vaccine production.

4.6 Solid lipid nanoparticles

Solid lipid NPs (SLNs) have been extensively studied over the

past decade because they provide the combinatorial eﬀect of

various carrier systems, including polymeric nanoparticles,

emulsions, and liposomes.

239

SLNs have changed the dimen-

sions of gene and drug delivery because of their advantages,

including biocompatibility and safety profile, avoidance of

organic solvents, ease of large-scale synthesis, water-based

technology, physicochemical stability, ability to encapsulate

proteins and nucleic acids along with hydrophilic, hydro-

phobic drugs and bioactive compounds, and controlled and

sustained release of loaded components.

240–242

SLNs are

spherical nanoparticles with diameters of 50–1000 nm.

239,242

SLNs remain solid at room temperature because of the core of

SLNs. The core of SLNs is composed of solid lipids such as

glycerides, steroids, fatty acids, or waxes, whereas LNPs com-

prise solid and liquid crystalline lipids.

201,243

SLNs are

superior to lipid nanoparticles in protecting nucleic acids

Fig. 7 (a) Schematic of LNP synthesis, including the components used

to make LNPs via microﬂuidic mixing and the expected resulting struc-

ture. (b) Visualization of the design process used to generate libraries A

and B with library A resulting from orthogonal DOE screening of a wide

range of excipient molar ratios, and library B examining more formu-

lations within a narrowed range of excipient ratios based on the results

from the library A screen. (c) Schematic of the CAR T cell production uti-

lizing either LNPs or EP for mRNA delivery to T cells. The treated T cell

populations may diﬀer in viability and CAR potency depending on the

transfection method. Still, both can generate functional CAR T cells to

induce targeted cancer cell killing. Adapted with permission from ref.

RSC Pharmaceutics Review

from degradation and leakage during storage. SLNs are syn-

thesized using physiological, biodegradable, and biocompati-

ble lipids in a solid state at room temperature, emulsifiers, or

a combination of pharmaceutical agents and solvents.

Diﬀerent methods have been extensively investigated for the

synthesis of SLNs, such as emulsion-solvent evaporation,

244

microemulsion,

245

high-shear homogenization,

246

ultra-

sonication,

247

solvent injection methods,

248

nanoprecipitation

(solvent displacement technique) and so on.

193

SLNs possess a

solid lipid core loaded with the active ingredient and stabilized

with an outer surface of surfactant. Nonviral delivery of

plasmid DNA was eﬃciently done using SLP-based cargos.

249

The key ingredients of the SLNs prepared via the solvent-emul-

sification method include cholesteryl oleate glycerol trioleate,

and DOPE cholesterol3β-[N-(dimethylaminoethane)carbamoyl

(DC)-cholesterol]. Furthermore, plasmid DNA was incorporated

into SLNPs. In vitro studies suggested improved transfection

eﬃciency of plasmid DNA in dendritic cells. Song et al. (2017)

synthesized cationic solid lipid NPs (CSLNs) for the delivery of

siRNA and β-NaYF

:Yb,Er upconversion nanoparticles (UCNPs)

and applied these for bioimaging and gene therapy in A549

cells.

194

CSLPs were synthesized via the thin-film dispersion

method using a peptide-based cationic lipid, CDO14, and

further conjugated to UCNPs and siRNA by electrostatic inter-

action. Gene silencing, bioimaging, and cytocompatibility

studies demonstrated that encapsulated siRNA and UCNPs

could eﬃciently be delivered and internalized into the cells

and displayed superior cellular imaging and gene silencing

with CSLPs (Fig. 8a & b). Cytotoxicity studies performed on

A549 cells with an incubation of 48 h showed that CLSPs were

cytocompatible, with cell viability higher than 90% (Fig. 8c).

4.7 Commercial history of lipid-based drug delivery agents

The commercial history of lipidic formulations for drug and

gene delivery can be traced back to the late 20th century when

the first liposomal drug, Doxil (doxorubicin hydrochloride

liposome injection), was approved by the US FDA in 1995.

250

Since then, lipid formulations have become a popular and

eﬀective means of delivering a wide range of therapeutic

agents, including cancer drugs, anti-inflammatory agents, and

nucleic acids for gene therapy. Liposomes, LNPs, and spherical

structures can encapsulate drugs and protect them from rapid

clearance and degradation, enabling targeted delivery to cells

and tissues. The market for lipid-based drug delivery systems

has snowballed in recent years, with several new drugs and

gene therapies using lipidic formulations in development and

commercialization. One of the main advantages of lipidic for-

mulations is their ability to enhance the eﬃcacy of drugs and

reduce their toxic side eﬀects. All these advantages have led to

the approval of several lipid-based drugs for the treatment of

cancer, including Doxil,

250

Camptosar (lipid NPs),

200

and

DaunoXome (daunorubicin citrate liposome injection).

251

recent years, the use of lipidic formulations for gene delivery

has also become increasingly popular, leading to the develop-

Fig. 8 (a) Uptake of CSLNs into A549 cells. The ﬂuorescence microscope images after treatment with CSLNs (excited by 980 nm light) at concen-

trations of (a) 5 μgμL

−1

, (b) 10 μgμL

−1

, (c) 15 μgμL

−1

and (d) 20 μgμL

−1

. (b) Gene silencing mediated by CSLNs. (c) Cytotoxicity of CSLNs/siRNA and

Review RSC Pharmaceutics

ment of new treatments for genetic diseases and other dis-

orders. During the outbreak of SARS-CoV-2, several LNP mRNA

vaccines were developed to fight the disease, such as

Comirnaty and Spikevax.

177,252

Lipid-based delivery systems

are widely accepted in clinical applications and have been

approved by the FDA to deliver various therapeutics, including

drugs and nucleic acids (Table 4).

5. Nanomaterial delivery vehicles for

nucleic acids: promise and challenges

Nucleic acids are widely explored for the treatment of a broad

spectrum of diseases including cancer, heart diseases, genetic

disorders, and viral and bacterial infections. However, the

applicability of nucleic acid in therapeutics is challenging due

to enzymatic degradation, and unfavorable physiocochemical

properties (hydrophilicity and high molecular weight, negative

charge) resulting in reduced cellular uptake, poor in vivo stabi-

lity, rapid excretion or uptake by non-targeted cells.

72,255

Suitable vectors that can overcome inadequate eﬃcacy, suscep-

tibility to enzymatic degradation, low bioavailability, and oﬀ-

target side eﬀects are required to increase the therapeutic

eﬃcacy of nucleic acids. Nanomaterials oﬀer exciting possibili-

ties for the therapeutic delivery of nucleic acids, like DNA or

RNA.

223

These nanocarriers hold immense potential for the

pharmaceutical landscape as they revolutionized gene therapy,

RNA interference, and vaccine development. Compared with

traditional methods of delivering nucleic acids into cells,

nanomaterials oﬀer a multitude of advantages. (1) Nucleic

acids on their own have diﬃculty penetrating cell membranes

and reaching their target sites. Nanomaterials can encapsulate

and protect nucleic acids from enzymatic degradation, facili-

tating their cellular uptake and delivery across biological bar-

riers.

234

(2) Nanocarriers can be engineered with specific mole-

cules on their surface that recognize and bind to particular cell

types. This targeted delivery allows researchers to focus the

eﬀects of nucleic acids on the desired cells, reducing side

eﬀects on healthy tissues.

249

(3) Nanocarriers can be designed

to release their nucleic acid cargo in a controlled manner over

time. This sustained release can be crucial for some therapies,

allowing for longer-lasting eﬀects.

256

(4) Some nanocarriers

can be engineered to combine functionalities like delivering

therapeutic molecules and imaging capabilities, oﬀering a

more comprehensive approach to treatment and monitor-

ing.

257

(5) Nanocarriers can be designed to release their cargo

in response to specific stimuli like pH changes or light

exposure.

237

This allows for a more controlled and localized

release of nucleic acids within the body.

While nanomaterials oﬀer exciting possibilities for nucleic

acid delivery, there are also some limitations and potential

downsides to consider. (1) Despite targeting strategies, there is

a risk that nanocarriers could deliver nucleic acids to unin-

tended cell types. This can lead to unwanted side eﬀects or

even toxicity.

(2) The body’s immune system may recognize

nanocarriers as foreign invaders and trigger an inflammatory

response. This can limit the eﬀectiveness of the therapy or

cause complications.

258

(3) Developing and manufacturing

safe and eﬀective nanocarriers for nucleic acid delivery can be

complex and expensive. This can hinder the accessibility and

aﬀordability of these therapies. (4) Some nanomaterials may

not be eﬃciently cleared from the body after delivering their

payload. This can raise concerns about potential long-term

eﬀects.

259

Table 4 Overview of the FDA-approved lipid-based therapeutics

no. Commercial

name Developer Date of

approval Lipid Drug Treatment Ref.

1 Diprivan Crucell Feb 23, 1989 Liposome Propofol Induction and maintenance of

anesthesia 251

2 Doxil® Janssen Nov 17, 1995 Liposome Dox Ovarian and breast cancer 250

3 Daunoxome® Galen Apr 8, 1996 Liposome Daunorubicin HIV-associated Kaposi’s sarcoma 251

4 AmBisome Gilead Sciences Nov 8, 1997 Liposome Amphotericin

BInvasive fungal infections 251

5 Cytosar-U® Pfizer Jan 8, 1999 Lipid NPs Cytarabine Leukemia and lymphoma 200

6 Visudyne Bausch and Lomb Apr 30, 2002 Liposomal Verteporfin Macular degeneration, pathologic, or

ocular histoplasmosis 251

7 Camptosar® Pfizer June 24, 2004 Lipid NPs Irinotecan Metastatic colon or rectum cancer 200

8 Vinlon™Talon Therapeutics,

Inc. Sept 8, 2012 Liposome Vincristine Acute lymphoblastic leukemia 200

9 Onivyde® Merrimack Oct 22, 2015 Liposome Irinotecan Various metastatic cancers, including

breast, pancreatic, sarcomas, or brain 251

10 Nocita® Aratana therapeutics August 12, 2016 Liposome Bupivacaine Anesthetic 253

11 Vyxeos

CPX-351® Jazz Pharmaceuticals Aug 3, 2017 Liposome Cytarabine :

daunorubicin Acute myeloid leukemia 251

12 Onpattro®

(patisiran) Alnylam

Pharmaceuticals Inc. Aug 8, 2018 Lipid NPs siRNA Hereditary transthyretin amyloidosis 235

13 Givosiran

(Givlaari®) Alnylam

Pharmaceuticals Inc. Nov 20, 2019 Lipid NPs siRNA Hepatic porphyria 254

14 Comirnaty® BioNTech, Pfizer Aug 23, 2021 Lipid NPs mRNA COVID-19 177

15 Spikevax® Moderna Jan 31, 2022 Lipid NPs mRNA COVID-19 252

RSC Pharmaceutics Review

6. Future strategies

Researchers are actively exploring several strategies to further

improve nanomaterial-based delivery vehicles for nucleic

acids, addressing the current limitations and unlocking their

full potential. (1) Attaching targeting ligands to the surface of

nanocarriers can significantly improve their specificity. These

ligands can bind to receptors present on specific cell types,

directing the delivery of nucleic acids to the desired location.

(2) Developing nanocarriers that respond to specific environ-

mental cues within the body. For example, carriers could

release their cargo in response to changes in pH or tempera-

ture, allowing for targeted release at the disease site.

260

(3)

Modifying the surface properties of nanocarriers to minimize

interactions with the immune system and reduce the risk of

inflammatory responses.

261

This can involve using biocompati-

ble coatings or manipulating surface charges. (4) Developing

nanocarriers from biodegradable polymers that are naturally

broken down and eliminated by the body, reducing long-term

accumulation concerns.

259

(4) Optimizing the rate at which

nucleic acids are released from nanocarriers. This can help to

minimize oﬀ-target eﬀects and improve the overall therapeutic

eﬃcacy. (5) Developing nanocarriers that can co-deliver nucleic

acids with other therapeutic agents, such as drugs or imaging

molecules, for combination therapy and improved treatment

outcomes.

262

(6) Creating nanocarriers that can be tracked

within the body using imaging techniques, allowing research-

ers to monitor their delivery pathway and optimize targeting

strategies. (7) Developing eﬃcient and cost-eﬀective pro-

duction methods for well-defined and consistent nanocarriers,

facilitating their wider clinical application.

263

By addressing

these challenges and focusing on these future strategies,

researchers can unlock the full potential of nanomaterial-

based delivery vehicles for nucleic acids. This has the potential

to revolutionize how we treat various diseases, oﬀering more

targeted, eﬀective, and safer therapies based on gene therapy,

RNAi, and DNA vaccines.

7. Conclusion

Nucleic acid-based therapeutics has experienced the trans-

formation from concept into clinical reality in recent years.

The first FDA-approved siRNA-based drug, Onpattro (2018),

provides a platform for further developing NA therapeutics to

cure rare conditions, cancers, and infectious diseases.

235,264

Since then, significant progress has been made to improve

their therapeutic eﬀect, further amplified in the development

of the SARS-Cov-2 vaccine worldwide.

157

However, clinical

availability usually requires modification of NAs and their car-

riers to increase nuclease resistance and enhance cellular

uptake. SNAs with densely packed 3D structures have overcome

some challenges without further modification. SNAs have

minimized the nuclease degradation of delivered NAs.

191

Despite this, significant issues still need to be addressed, most

notably in eﬀectively providing therapeutic NAs to the targeted

site in the required quantity. The stability of SNA structures

can be improved by tuning them with other NPs, resulting in

tissue/organ-specific delivery. Among all the classes of delivery

agents, lipid-based nanocomplexes have shown ideal delivery

eﬃciency at the clinical level.

265

The clinical trials of the

diﬀerent diseases have proved the importance of lipid-based

NPs in NA therapeutics. Over the past decade, several NA thera-

peutics have been in clinical trials, and only a few enjoyed

commercial successes. These points highlight the challenges

in translating new drugs from animals to humans. With the

success story of Onpattro, lipid-based technology is accepted

worldwide and is ready to uplift the next wave of NAs thera-

peutics for vaccination, gene therapy, and protein production.

Undoubtedly, the field of NAs is undergoing a massive expan-

sion, and their potential applications in the treatment of

chronic disorders, immunotherapy, and personalized medi-

cine will ensure their development to revolutionize the bio-

medical field in the near future.

Abbreviations

DOTAP 1,2-Dioleoyl-3-(trimethylammonium) propane

ASO Antisense oligonucleotides

CNMs Carbon nanomaterials

CLs Cationic lipids

FDA Food and drug administration

AuNPs Gold NPs

GFP Green fluorescent protein

ILs Ionizable lipids

IONPs Iron oxide NPs

LNPs Lipid NPs

mRNA Messenger RNA

miRNA MicroRNA

NPs Nanoparticles

NAs Nucleic acids

pDNA Plasmid DNA

PAMAM Poly(amidoamine)

PEG Polyethylene glycol

PEG-DSPE Polyethylene-glycol-2000-1,2-distearyl-3-sn-

phosphatidylethanolamine

PEI Polyethyleneimine

PLGA Polylactide-co-glycolide

PNPs Polymeric NPs

QDs Quantum dots

siRNA Short interfering RNA

SiNPs Silicon NPs

SLNPs Solid lipid NPs

SNAs Spherical nucleic acids

UCNPs Upconversion nanoparticles

ZILs Zwitterionic lipids.

Author contributions

All the authors read and approved the manuscript.

Review RSC Pharmaceutics

Data availability

No primary research results, software or code has been

included and no new data were generated or analysed as part

of this review.

Conﬂicts of interest

The authors declare that they have no competing interests.

Acknowledgements

This project has received funding from the European Union’s

Horizon Europe research and innovation program under Marie

Skłodowska-Curie grant agreement No. 101103113. M. J.

Mehta was supported by the National Research Foundation of

Korea grant (No. 2021R1A2C1007668 and RS-2024-00398030)

funded by the Korean government.

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Full-text available

Mar 2023

The viral infection spreads with the assistance of a host. Traditional antiviral therapies cannot provide long-term immunity against emerging and drug-resistant viral infections. Immunotherapy has evolved as an efficient approach for disease prevention and treatment, which include cancer, infections, inflammatory, and immune disorders. Immunomodulatory nanosystems can dramatically enhance therapeutic outcomes by combating many therapeutic challenges, such as poor immune stimulation and off-target adverse effects. Recently, immunomodulatory nanosystems have emerged as a potent antiviral strategy to intercept viral infections effectively. This review introduces major viral infections with their primary symptoms, route of transmission & targeted organ, and different stages of the viral life cycle with respective traditional blockers. The IMNs have an exceptional capacity for precisely modulating the immune system for therapeutic applications. The nano sized immunomodulatory systems permit the immune cells to interact with infectious agents enhancing lymphatic drainage and endocytosis by the over-reactive immune cells in the infected areas. Immune cells that can be modulated upon viral infection via various immunomodulatory nanosystems have been discussed. Advancement in theranostics can yield an accurate diagnosis, adequate treatment, and real-time screening of viral infections. Nanosystem-based drug delivery can continue to thrive in diagnosing, treating, and preventing viral infections. The curative medicine for remerging and drug-resistant viruses remains challenging, though certain systems have expanded our perception and initiated a new research domain in antiviral treatments.

Recent Progress in the Endosomal Escape Mechanism and Chemical Structures of Polycations for Nucleic Acid Delivery

Article

Jan 2024
MACROMOL BIOSCI

Nucleic acid‐based therapies are seeing a spiralling surge. Stimuli‐responsive polymers, especially pH‐responsive ones, are gaining widespread attention because of their ability to efficiently deliver nucleic acids. These polymers can be synthesized and modified according to target requirements, such as delivery sites and the nature of nucleic acids. In this regard, the endosomal escape mechanism of polymer–nucleic acid complexes (polyplexes) remains a topic of considerable interest owing to various plausible escape mechanisms. This review describes current progress in the endosomal escape mechanism of polyplexes and state‐of‐the‐art chemical designs for pH‐responsive polymers. We also discuss the importance of the acid dissociation constant (i.e., p K a ) in designing the new generation of pH‐responsive polymers, along with assays to monitor and quantify the endosomal escape behavior. Further, we address the use of machine learning in p K a prediction and polymer design to find novel chemical structures for pH responsiveness. This review will facilitate the design of new pH‐responsive polymers for advanced and efficient nucleic acid delivery. This article is protected by copyright. All rights reserved

Recent progress on nanosystems for nucleic acid delivery

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