Research Progress on Liposome Pulmonary Delivery of Mycobacterium tuberculosis Nucleic Acid Vaccine and Its Mechanism of Action

Abstract

Traditional vaccines have played an important role in the prevention and treatment of infectious diseases, but they still have problems such as low immunogenicity, poor stability, and difficulty in inducing lasting immune responses. In recent years, the nucleic acid vaccine has emerged as a relatively cheap and safe new vaccine. Compared with traditional vaccines, nucleic acid vaccine has some unique advantages, such as easy production and storage, scalability, and consistency between batches. However, the direct administration of naked nucleic acid vaccine is not ideal, and safer and more effective vaccine delivery systems are needed. With the rapid development of nanocarrier technology, the combination of gene therapy and nanodelivery systems has broadened the therapeutic application of molecular biology and the medical application of biological nanomaterials. Nanoparticles can be used as potential drug-delivery vehicles for the treatment of hereditary and infectious diseases. In addition, due to the advantages of lung immunity, such as rapid onset of action, good efficacy, and reduced adverse reactions, pulmonary delivery of nucleic acid vaccine has become a hot spot in the field of research. In recent years, lipid nanocarriers have become safe, efficient, and ideal materials for vaccine delivery due to their unique physical and chemical properties, which can effectively reduce the toxic side effects of drugs and achieve the effect of slow release and controlled release, and there have been a large number of studies using lipid nanocarriers to efficiently deliver target components into the body. Based on the delivery of tuberculosis (TB) nucleic acid vaccine by lipid carrier, this article systematically reviews the advantages and mechanism of liposomes as a nucleic acid vaccine delivery carrier, so as to lay a solid foundation for the faster and more effective development of new anti-TB vaccine delivery systems in the future.

Background

According to statistics, Mycobacterium tuberculosis (MTB) is one of the leading causes of death from infection worldwide,¹ ranking 13th globally.² The World Health Organization (WHO) has released the latest Global Tuberculosis Report 2022,³ which shows that there were 10.6 million (95% confidence interval: 9.9–11 million) new tuberculosis (TB) patients worldwide in 2021, including 6 million adult males, 3.4 million adult females, and 1.2 million children. Of these, 6.7% were infected with HIV.

The development of vaccines is one of the most important advances in modern medicine. Prevention is the main means to control infectious diseases, and vaccination is considered to be the most effective measure. The only currently recognized effective vaccine against TB is the Bacillus Calmette Guerin (BCG), which was attenuated from the Mycobacterium tuberculosis bovis. However, BCG can only provide protection against severe TB in children and adolescents, and its effect is variable against pulmonary TB in adults.⁴ In addition, as a live attenuated vaccine, BCG can also cause local or systemic infection in immunocompromised populations, such as HIV-positive individuals at high risk for active TB disease.⁵ Therefore, there is an urgent need to develop a more effective, long-lasting, safe, and cost-effective new TB vaccine.

Different from traditional protein-based vaccines, nucleic acid vaccines are vaccines based on pathogen gene sequences, which is the third generation of vaccines after traditional vaccines (including inactivated vaccines and attenuated live vaccines) and recombinant genetic engineering vaccines (including genetic engineering subunit vaccines, synthetic peptide vaccines, and recombinant genetic engineering live vaccines). They can express antigen proteins stably in the host for a long time, and the immune response is only for the specific antigen of concern. Therefore, the immunogenicity produced by nucleic acid vaccines is more targeted.^6,7 It can not only induce humoral immune responses but also induce a high level of cellular immune response, especially cytotoxic T lymphocyte (CTL), which can recognize, kill, and destroy infected cells and eliminate intracellular pathogens.⁸ This is of great significance for the elimination of MTB that lives in macrophages.⁹

In addition, DNA vaccines have not been found to have any adverse reactions in animal and human experiments, especially there is no evidence to suggest that they will produce autoimmunity or plasmid DNA (pDNA) integration into chromosomes, which has good safety and tolerance.^10,11 However, nucleic acid vaccines have performed poorly in large animal and human clinical trials, and the results of human trials have been disappointing.^12–14 This may be related to the lack of effective delivery, which affects its clinical application.¹⁵

The immune pathway is an important factor affecting antigen immunogenicity and immune effect, so different routes of vaccination and administration will also greatly affect the efficacy and immune effect of the vaccine.¹⁶ Common routes of vaccination include skin scratches, intradermal injection, subcutaneous injection, intramuscular injection, oral administration, nasal spray, nebulized inhalation, etc. These immunization modalities can induce systemic and local immune responses.¹⁷ To date, the conventional methods of nucleic acid vaccination have been intramuscular or intradermal injections,¹⁸in addition to oral,¹⁹ intranasal,²⁰ intravenous injection,²¹ and other routes to deliver immunogens.

The most commonly used traditional route of administration is intramuscular, but this route of administration is invasive and causes pain or discomfort at the injection site, with a risk of local inflammation or infection. Moreover, the vaccine is absorbed from the muscle tissue into the blood before being distributed to the target site, so it takes more time to reach a steady state, leading to persistent effects even after stopping vaccination.²² In addition, Sokolova et al. conducted a clinical study comparing the intramuscular and inhalation routes, and the results showed that both routes could produce good immune levels.²³ However, our study showed that TB DNA vaccines immunized by inhalation routes can induce local lung immunity earlier than intramuscular injection routes (results have not yet been published). Oral routes are also attractive for vaccination, but oral administration of subunit vaccines or inactivated vaccines needs to avoid the drawbacks of being degraded in the gastrointestinal tract and relatively low immunogenicity, especially nucleic acid vaccines are easily degraded in the body and need to be delivered by protective particles.²⁴

Studies have confirmed that intravenous administration could only induce poor mucosal immunity, and the effect of administration was limited.²⁵ Therefore, there is a growing need for better delivery routes to improve the stability and efficacy of vaccines.

The lungs have a large surface area, thin alveolar epithelium, and an extensive vascular system, which can allow rapid and efficient absorption of active ingredients in the lungs, providing advantages for the systemic delivery of vaccines and other drugs.²⁶ The lungs have natural permeability to proteins. Due to the fact that the presence of drug efflux transporters and metabolic enzymes in the lungs is much lower than in the gastrointestinal (GI) tract, small-molecule inhalation is also a rapid way of entering the body relative to the gastrointestinal tract.²⁷ However, the absorption time of lipophilic small molecules in the lungs was significantly shorter than that of water-soluble small molecules.^28,29 Vaccine lung delivery is not only a noninvasive delivery mode but also directly delivered to lung tissue through natural respiratory infection pathways, obtaining higher local concentrations, enhancing lung local immunity, and improving lung local protection.^30,31

Due to stability, solubility, and nonspecific toxicity, some promising small-molecule and gene vaccines are considered to have poor effects on direct delivery in vivo.^32,33 At present, different delivery methods studied mainly include chemical methods (such as nanoparticles and liposomes), physical methods (such as electroporation,³⁴ gene guns, and microneedle arrays), biological methods (such as bacterial and viral vectors), etc.^35–38 In this article, we review the research progress on the immunoprotective effect and mechanism of TB nucleic acid vaccine-coupled lipid vector delivered through the lungs.

Liposomes and Their Application Advantages as Vaccine Carriers

Liposomes

The first closed bilayer phospholipid system known as liposome was first discovered by Bangham and colleagues in the 1960s and was soon investigated as a potential drug delivery system.^39,40 Current common nonviral nanoparticle gene delivery systems are shown in Table 1. Compared with other types of carriers, liposomes and their derivatives are widely used as carriers to improve drug efficacy. Liposomes are microvesicular vesicles that encapsulate drugs in lipid-like bilayers. They can be good adjuvants for lung delivery because the lung is mainly composed of a large number of alveoli, and lipids are an important part of alveolar surfactant, among which phospholipids are the most abundant type.⁴¹ It is one of the most successful inhalable nanocarriers.

Table 1.

Nonviral Nanoparticle Gene Delivery System

Carrier type	Application areas	Carrier material	Example
Peptides or peptide carriers	Precise treatment of major diseases and cancer	Cationic polypeptides containing lysine, histidine, and arginine, various protein components	^157,158
Exosomes	Small-molecule chemical drugs and gene drugs are used to treat tumors and Alzheimer's disease	Rich in selected proteins, lipids, nucleic acids, and sugar dropouts	¹⁵⁹
Liposomes and their derivatives
LP	Immunoprophylaxis, gene therapy	Phospholipid Cholesterol Polyethylene glycol lipids	¹⁰⁸
CNE	Infectious disease field		¹⁶⁰
SLN	Immunoprophylaxis, gene therapy		¹⁶¹
LPP	Immunoprophylaxis, gene therapy		¹⁶²
LNP	Infectious diseases, protein replacement therapy, metabolic diseases, genetic diseases, cancer, and other treatments	Ionizing and cationic lipids cholesterol phospholipid Polyethylene glycol lipids	¹⁶³
Polymer nanodrug carriers
Chitosan	Cancer treatment Treatment of metabolic diseases Antimicrobial field	Polysaccharides (such as chitosan) PLA block polymers, etc.	^164–168
PLGA
PLA
Polymer micelles
Dendritic molecules
Inorganic nanoparticles	Diagnosis, treatment, imaging	Lipids, precious metal nanoparticles, rare earth metals, some semiconductor materials	¹⁶⁹

CNE, cationic nanoemulsion; LNP, lipid nanoparticle; LP, liposome complex; LPP, liposomal polymer; PLA, polylactic acid; PLGA, polylactic acid-co-polyglycolic acid; SLN, solid lipid nanoparticle.

At present, several electrically neutral lipids, such as cholesterol, phosphatidylcholine (PC), diolylphosphatidylethanolamine (DOPE), and alkyl phosphatidylcholine (APC), have been used as auxiliary lipids to prepare new multicomponent cationic lipid preparations as gene delivery carriers.⁴² The biophysical and chemical properties of nanoparticles determine their in vivo biocompatibility in the field of nanotherapeutics, and hydrophobicity, size, and surface charge are the main parameters affecting the biocompatibility of nanoparticles.⁴³ Compared with neutral or anionic lipids, the positive charge on the surface of cationic lipids is beneficial to the fusion with cells in vivo, but it also inevitably increases the immunogenicity and cytotoxicity in vivo and is more likely to induce hemolysis and platelet aggregation.^44–47 In the past 50 years, based on the early pioneering work of Schreier et al. on the pulmonary application of liposomes, liposomal aerosols are thought to be able to play an important role in the treatment of lung diseases, including intracellular infections, immune disorders, and genetic defects. The study also illustrates a targeted strategy for selectively delivering drugs to phagocytic (alveolar macrophages) and nonphagocytic (epithelial) cells that are infected or damaged in the lung.⁴⁸ Since then, numerous liposome researchers have further expanded and extended the application of liposomes in the lung,⁴⁹ such as the delivery of anticancer drugs,⁵⁰ antibacterial drugs,⁵¹ gene delivery,⁵² and other fields have entered the stage of clinical trials. It has been proven to be an effective and safe preventive vaccine and therapeutic delivery vector.^53,54

Among the recently FDA-approved SARS-CoV-2 messenger RNA (mRNA) vaccines, lipid-based nanoparticles have been shown to be well suited for antigen presentation and enhanced immune stimulation to elicit effective humoral and cellular immune responses.^55,56 Compared with other vaccine adjuvants, liposomes have many unique advantages: (1) they are biocompatible, and biodegradable, with low toxicity and antigenicity⁵⁷; (2) sustaining release and controlling release; (3) they are adjustable targeting; (4) they are well tolerated in the lungs, especially liposomes prepared with phospholipids and pulmonary lipids such as dipalmitoyl phosphatidylcholine (DPPC)⁵⁸; and (5) they slow down the chemical and biological degradation of nucleic acid in vivo and so widely used.^59–62 The modified liposomes, which allow for longer circulation times or more targeted delivery to the therapeutic target, enhance the immunoprotection of vaccines and show no detectable toxicity in vivo.^63,64

Based on this advantage, in the field of vaccine development, liposomes and their derivatives have received great attention and become the hot spot of current research.^65,66

Lipid vector-based nucleic acid vaccines

When nucleic acid vaccines are injected alone, their immunogenicity is poor. The naked plasmids are difficult to enter the cells to play a role due to their large molecular weight, low biological stability, relatively single function, and easy to be decomposed under the action of low pH and enzymes in the body. Therefore, the immunogenicity of the nucleic acid vaccine in the host will be affected by many factors, which limits their clinical application.^67,68

At present, many liposomes and their derivatives have been marketed.⁶⁹ Many complex liposome formulations are still under development, and some of these lipid drug-loading systems have entered clinical trials.^70,71 More recently, the liposomal inhaled Arikayce^® was approved by the FDA in 2018 for the treatment of the Mycobacterium avium complex (MAC) lung disease as part of a combination antibacterial drug regimen for adult patients who have limited or no alternative treatment options (Arikayce; Insmed, Incorporated).^72,73 Transpulmonary inhalation of ciprofloxacin liposome (Pulmaquin) to optimize the treatment of pulmonary infections in patients with noncystic fibrosis (CF) bronchiectasis has also entered phase III clinical trials. In 2019, VanDevanter et al.⁷⁴ used ARD-3150 (inhaled liposomal ciprofloxacin) to treat patients with non-CF bronchiectasis and chronic Pseudomonas aeruginosa lung infection for 48 weeks, and showed a reduction in microbiological titer during the treatment periods, with a decrease in ciprofloxacin susceptibility that recovered during the off-treatment periods.

Subsequently, Chalmers et al.⁷⁵ found that inhaled ARD-3150 in patients with bronchiectasis and chronic Pseudomonas aeruginosa infection significantly improved respiratory symptoms during the treatment, while these improvements disappeared during discontinuation. Liposomes have the advantage that the excipient is rapidly cleared by a natural catabolism process. However, few liposomal vaccines have been approved for marketing (Table 2).^76,77 The antigens coused with liposomes include proteins,⁷⁸ peptides,⁷⁹ RNA,⁸⁰ and DNA.⁸¹ These further demonstrate the potential of liposomes as multifunctional vaccine delivery carriers.⁸²

Table 2.

Liposomal Vaccines

Name	Indications	State	Year
Epaxal^®	Hepatitis A vaccine	Approved for marketing	1994
Inflexal V^®	Flu vaccine	Approved for marketing	1997
Mosquirix^®	Malaria vaccine	Approved for marketing	2015
Shingrix^®	Recombinant herpes zoster virus vaccine	Approved for marketing	2018

Application of liposomes in the delivery of different nucleic acids

The transfection efficiency of the lipid vector may be affected by the gene delivered. For example, pDNA can be delivered into cells more efficiently than linear DNA because pDNA produces a lower effective negative charge than linear DNA. pDNA has attracted much attention in the field of gene therapy because of its safety and stability, large tolerance to a variety of foreign genes, easy production, and no direct adverse reactions in the human immune system. Importantly, unlike mRNA, pDNA needs to reach the nucleus and not just the cytoplasm for successful translation of proteins. It is a common method to select biocompatible amino acids as lipid materials in the preparation of cationic liposomes, and the selection of cationic liposomes containing DOPE can improve the transport of pDNA in vivo.⁸³

Pulmonary therapies based on cationic lipids have shown the potential to treat various pulmonary and genetic disorders. As a novel lipid-based nanosystem, nanostructured lipid carriers (NLC) can be used for local or targeted drug delivery.⁸⁴ Among them, the inhalation route was explored for site-specific drug delivery and toxicity reduction in lung cancer,⁸⁵ However, due to the use of mainly cationic lipids or positive charges, inherent cytotoxicity or nanotoxicity may still exist.⁸⁶ The transferrin (Tf)-modified NLC has achieved the codelivery of anticancer drugs and DNA in the targeted therapy of lung cancer, and has shown high gene transfection and enhanced antitumor activity in in vitro and in vivo studies, with a focus on low cytotoxicity.⁸⁷

mRNA is a natural product of genes. Its main role in the cells is to carry genetic information and serve as a direct template for protein synthesis. It has the characteristics of a subunit vaccine and attenuated live vector and can induce humoral and cellular immunity at the same time.⁸⁸ mRNA is single-stranded, structurally unstable, and easily decomposed by nucleases. It is a hydrophilic polymer nucleic acid rich in anion groups, which is not easy to pass through the lipid bilayer cell membrane. Lipid nanoparticle (LNP) delivery technology can protect mRNA, help them enter human cells, and assist them in producing protein antigens in cells.

The outbreak and pandemic of COVID-19 have greatly promoted the application of LNP technology and the marketing process of mRNA vaccines. The mRNA vaccines encoding the SARS-CoV-2 spike protein antigen and delivered to host cells by LNP technology have been successfully licensed and have shown good safety and efficacy.^89,90 At present, the clinical research on mRNA mainly focuses on vaccination, protein replacement therapy, and the treatment of genetic diseases.⁹¹

Small interfering RNA (siRNA) has been extensively studied worldwide due to its advantages such as gene silencing specificity, easy synthesis, and short development cycle.⁹² It can inhibit specific genes that cause viral infection and cancer. Compared with pDNA, siRNA has a lower molecular weight and consists of only 20–30 base pairs. It does not need to be delivered to the nucleus for effect, and siRNA is more specific than microRNA (miR). However, its delivery is relatively difficult, and the main problems it faces are as follows: (1) siRNA is extremely unstable, due to the degradation of nucleases and the half-life of siRNA is very short; (2) it is difficult for siRNA to enter the target site and needs to pass through many barriers in the human body; and (3) siRNA administration has poor targeting and is prone to off-target effects, causing unnecessary toxic biological effects in nontarget tissues and cells. Therefore, cationic lipids and lipid analogues become good delivery carriers for siRNA, providing stability and targeting for delivery.⁹³ In 2018, Onpattro (patisiran), developed by Alnylam, was the first RNA interference (RNAi) drug approved for market by the US Food and Drug Administration. It is an RNAi therapy that uses LNP technology to deliver the drug directly to the liver for the treatment of hereditary transthyretin-mediated amyloidosis (hATTR).^94–96

MicroRNAs are also a class of endogenous noncoding small RNA molecules that have the function of regulating gene expression. They have become potential therapeutic drugs for various diseases by inhibiting mRNA translation and regulating other functional genes, and are gradually being applied in gene therapy. Synthetic cationic liposomes have become a safe and efficient delivery vector for miR application in gene therapy. Maroof et al. prepared DOTAP/Chol/DOPE liposomes loaded with miR-34b-5p and effectively delivered miR-34b-5p to the sites of anaplastic thyroid cancer by intravenous injection, and found that miR-34b was overexpressed in anaplastic thyroid cancer cells, and the expression of vascular endothelial growth factor (VEGF-A) was inhibited both in vivo and in vitro. The results showed that miR-34b regulated the tumor-inhibitory properties of anaplastic thyroid cancer through VEGF-A.⁹⁷

Pulmonary Liposome Delivery of TB Vaccine

In recent years, liposomes as adjuvant delivery systems of the multifunctional vaccine have been applied to deliver various TB vaccines through different vaccination pathways, greatly improving the effectiveness of vaccine administration.

Liposome delivery of TB nucleic acid vaccine

The use of liposomes in TB nucleic acid vaccine formulations has shown promising results. Liposome wrapping can protect the DNA vaccine from the degradation of deoxyribonuclease and the disruptions of other physical and chemical mucosal barriers, promote absorption at the cellular level, and greatly improve the expression of DNA plasmids. Only a small amount of pDNA can induce effective immune responses. Therefore, the LNP technology appears to be a promising strategy for vaccine development.⁹⁸ However, the protective immune responses induced by different forms of nucleic acid vaccines through different immune pathways are not entirely the same. For example, intramuscular injection of DNA vaccines can induce systemic Th1-type immune responses, while the gene-gun skin injection mainly induces Th2-type immune responses. Oral vaccination can effectively induce systemic and mucosal immunity but deliver low doses (LD) to the lung.

After intravenous injection of liposomal vaccines, lipids are absorbed by reticulated endothelial cell-rich tissues (such as the liver and spleen), and then rapidly phagocytized and degraded by mononuclear phagocytes. Therefore, only about 25% of the dose administered intravenously can exert its effect on the lungs.⁹⁹

Oral administration of DNA vaccines is simple and easy to accept. The intestinal tract is considered the largest organ in the immune system, and effector cells are distributed throughout the entire intestinal mucosa. Through intestinal mucosal immunity, memory T and B cells can be induced and preferentially accumulate in the induced mucosal site. Therefore, the immune protection induced by oral vaccines is better than that of peripheral vaccination, and there are fewer adverse reactions. Wang et al.¹⁰⁰ encapsulated MTB pcDNA3.1 (+)/Ag85A DNA in liposomes and administered orally to immunize C57BL/6 mice. Significant expression of Ag85A protein was observed in the epithelium, microfolded cells (M cells), dendritic cells (DC), and Peyer's patches (pp) of the small intestine.

The levels of interleukin (IL)-2 and IFN-γ in intestinal epithelial lymphocytes (IELs) were significantly increased, while the levels of IL-4 were not significantly changed, the Ag85A-specific cytotoxicity of IELs was enhanced, and sIgA levels significantly increased. These data suggest that oral vaccination with liposome-encapsulated DNA vaccines can induce Th1-type cellular immune responses in the local intestinal mucosa, and the small intestinal epithelial cell compartments play a key role in modulating anti-TB immune responses to eliminate MTB, and can also induce antigen-specific mucosal cells and humoral immune responses. These findings have important implications for the design of new strategies for oral liposome TB DNA vaccines based on mucosal immunity to induce effective anti-TB immune responses.

Rosada et al.¹⁰¹ developed two novel MTB DNA-hsp65 vaccines entrapped (ENTR-hsp65) or complexed (COMP-hsp65) with nontoxic cationic liposomes EPC/DOPE/DOTAP (containing egg phosphatidylcholine [EPC], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE], and 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]), and intramuscular injection and intranasal delivery pathways were compared in mice. Although both liposome formulations induced a typical Th1-type immune response, the intramuscular route was not effective in preventing MTB infection in mice. In contrast, a single intranasal administration with 25 μg of COMP-hsp65 pDNA could significantly reduce colony-forming units (CFUs) in the lungs after the mice were attacked by Mtb, similar to the protective effect of four intramuscular injections with 100 μg of naked DNA-hsp65. This study suggests that a single administration of DNA vaccines through noninvasive nasal routes has significant advantages, as it can significantly reduce the vaccine dose.

D'Souza et al.¹⁰² design new methods to improve MTB Ag85 and PstS-3 DNA vaccines with two novel cationic and neutral lipid formulations, GAP-DLRIE:DOPE (aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine) and VC1052:DPyPE (aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide-diphytanoylphosphatidyl-ethanolamine, also known as Vaxfectin; Vical, Inc., San Diego, CA). Both formulations have previously been shown to enhance the antibody response to pDNA through nasal and muscular pathways.^103,104 Ag85A DNA formulated with Vaxfectin produced a 3- to 10-fold higher titer of Ag85A-specific antibody IgG and IgG isotypes in the serum of mice injected intramuscularly, and spleen T cells were stimulated by Ag85 antigen to produce Th1 cytokines (IL-2 and IFN-γ). The PstS-3 DNA in Vaxfectin can induce more persistent CD8⁺ T cell responses.

Intranasal immunization with Ag85A DNA in saline was ineffective, while intranasal immunization with DNA vaccine in GAP-DDLRIE:DOPE could induce the Th1-type cytokine response, but the response induced by intranasal immunization was significantly lower than that of intramuscular injection immunization with the same DNA dose. The combination of intramuscular and intranasal immunization with cationic lipids can produce stronger immune responses in the spleens, especially in the lungs. In addition, the Ag85B DNA formulated by Vaxfectin could reduce the bacterial count of spleens and lungs from the mice challenged intravenously with MTB H37Rv, enhancing protective efficacy. This study suggests that Vaxfectin may become an ideal adjuvant for intranasal immunization in DNA vaccines.

Rosada et al.¹⁰⁵ synthesized a peptide containing 21 amino acid residues (including SV40T nuclear localization signal, cation shuttle sequence, and C-terminal cysteamine group), which was then complexed with pDNA hsp65 and incorporated into cationic liposomes (composed of egg chicken L-α-phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-dioleoyl-3-trimethylammonium-propane [2:1:1 M]), forming a pseudoternary complex. The complex was immunized through an intranasal route to obtain a similar therapeutic effect on TB at a dose four times lower than that of naked DNA treatment, effectively reducing the therapeutic dose of DNA hsp65.

Okada et al.¹⁰⁶ developed a combined DNA vaccine (HSP65+IL-12/HVJ) expressing MTB HSP65 and IL-12 delivered by Japanese hemagglutinin virus (HVJ)-liposomes. The vaccine induced stronger CTL activity and significantly improved histopathological changes in mouse and guinea pig models after intramuscular injection compared with BCG, providing significant protective efficacy. In the cynomolgus TB model, the new vaccine also provides a higher level of protection than BCG. In addition, the combination of HSP65+IL-12/HVJ and BCG showed a synergistic protective effect in cynomolgus infected with MTB (100% survival rate).

Teng et al.¹⁰⁷ and Tian et al.¹⁰⁸ used dimethyldioctakylammonium (DDA) and two types of pattern recognition receptor agonists (monophospholipid A [MPLA] and artificially synthesized trehalose 6,6′-dibenate [TDB]) to form a liposome DDA/MPL/TDB (DMT) adjuvant, strongly inducing Th1-biased immune responses in immune mice. They incorporated two agonists (MPLA and TDB) into DDA, resulting in a more uniform liposome particle size, the aggregation between particles is reduced, storage stability is enhanced, and DNA-DMT releases DNA more slowly and persistently than DNA-DDA. They constructed a new eukaryotic expression plasmid pCMFO with the secretory expression of fusion protein CMFO (including four MTB antigens Rv2875, Rv3044, Rv2073c, and Rv0577), and its secreted expression was confirmed at the cellular level.

The pCMFO/DMT vaccine was administered intramuscularly, which induced the production of antigen-specific antibody IgG, high-level antigen-specific TNF-α and IFN-γ, and higher levels of CFMO-specific IL-2+TCM cells in the spleen of mice, significantly reduced the bacterial load in the spleen and lungs, and significantly alleviated pathological damage in the lungs. The short-term and long-term protective effects against MTB infection were consistent with that of BCG, suggesting that the PCMFO/DMT vaccine is a promising candidate for the TB vaccine.

Liposome delivery of other TB vaccines

Teng et al.¹⁰⁷constructed a fusion protein CTT3H based on five immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with MTB CD8⁺ T cell epitopes, and formed a CTT3H subunit vaccine also in the DMT liposome adjuvant, which immunized C57BL/6 mice by intramuscular injection. CTT3H/DMT vaccine not only induced antigen-specific CD4⁺Th1 response, but also increased the number of PPD- and CTT3H-specific IFN-γ⁺CD8⁺ T cells and elicited a strong CTL response to TB10.4, providing more effective protection than the phosphate buffer saline (PBS) control group and DMT adjuvant group.

In summary, liposomes can deliver TB nucleic acid vaccines and subunit vaccines through oral, intranasal, and intramuscular injection routes, effectively inducing anti-TB immunity, improving anti-TB protection efficacy, and greatly increasing the efficiency of vaccine vaccination. Liposomes, as vaccine adjuvant delivery systems, may solve the challenges of clinical application of nucleic acid vaccines.

Vaccine Lung Delivery, Lung Delivery Device, and Safety Evaluation

The natural infection route of MTB is through the respiratory tract, and pulmonary TB is its main disease type and source of transmission. When MTB enters the respiratory tract, it can be captured by the mucus in the nose, throat, trachea, and bronchi, eroded by enzymes, and discharged with the movement of cilia. However, when the body's defense ability is low and the protective function of the respiratory tract is weakened, MTB droplets will penetrate deep into the lower respiratory tract. Therefore, mucosal immunity will play an important role in resisting MTB infection.¹⁰⁹ In emerging research of inhalation immunity and inhalation therapy, small-molecule drugs in the form of solution, suspension, or dry powder can be quickly and efficiently absorbed from the lungs to achieve effective therapeutic concentrations due to rich pulmonary blood circulation, large absorption area, low degradation, high alveolar permeability, and short material exchange distances.

Moreover, the same therapeutic effect can be achieved at a lower dose of inhalation administered than that of oral or injection drugs. After being absorbed from the lungs, active ingredients can directly enter the bloodstream, avoiding the first-pass effect of the liver and improving bioavailability.⁹² This is very beneficial for drugs that are affected by systemic side effects, such as cancer drugs. Inhalation therapy may directly deliver high concentrations of active ingredients to the lungs, thereby maximizing the availability of drugs in the respiratory tract. Pulmonary administration is not only a noninvasive drug delivery method but also directly delivers targeted components to lung tissue, which can produce more specific immunotherapeutic effects for vaccines and other drugs in the local treatment of respiratory diseases.^93–95

Vaccine lung delivery

In recent years, increasing evidence has shown that mucosal immunity of vaccines produces a stronger immune response to lung infection sites than vaccination in the peripheral sites, providing better protection against respiratory MTB infection. For example, BCG is typically administered through an intradermal injection to prevent MTB in highly infected areas, but with only moderate protective efficiency. Mata et al.¹¹⁰ immunize mice with BCG vaccine through lung delivery, which can preactivate alveolar macrophages. After being attacked by MTB, the alveolar macrophages can effectively engulf and kill MTB, thereby limiting the early spread of MTB. When MTB attacks again, the alveolar macrophages can quickly reactivate and provide long-term protection. These results indicate that BCG lung delivery can induce well-trained innate memory-like responses in alveolar macrophages.

Counoupas et al.¹¹¹ used two Mtb antigens Ag85B and CysD (a component of the sulfur assimilation pathway) to construct a fusion protein and combined with the granular polysaccharide adjuvant Advax to construct a multistage TB mucosal vaccine CysVac2/Advax16. The vaccine delivered through intratracheal administration could induce the lung-resident antigen-specific memory CD4⁺T cells expressing IL-17 and ROR γ T (main transcriptional regulator of Th17 differentiation), and provide stronger protection against aerosol Mtb infection in mice compared with parenteral administration. Vaccines containing Advax have been proven to be safe and effective as inhaled formulations in human trials. Jeyanathan et al.¹¹² evaluated the safety and immunogenicity of human serotype 5 adenovirus vector vaccine AdHu5Ag85A through single-dose inhalation aerosol (including an LD aerosol group, a high-dose [HD] aerosol group) by Aeroneb Solo spray or intraperitoneal injection in Phase Ib clinical trials. Both vaccination routes were safe and well tolerated.

The vaccine injection group and LD aerosol group all induced Ag85A-specific T-cell responses in the blood. In addition, the LD group significantly induced the multifunctional memory CD4⁺ and CD8⁺ T cells in airway tissue residents, inducing sustained transcriptional changes in alveolar macrophages. This study is the first to demonstrate that nebulized inhalation of TB adenovirus vector vaccine is a safe and effective respiratory mucosal immunization method.

In conclusion, pulmonary delivery of TB vaccines follows the natural route of Mtb infection and can most closely mimic the immunity induced by Mtb in the respiratory tract, resulting in more comprehensive local lung immunity and systemic immunity. At present, direct pulmonary delivery is an ideal means of treatment and has become an important route for vaccine delivery.^113,114

Pulmonary delivery device and safety evaluation

Vaccine lung delivery can be achieved through direct targeting of the lungs. There are generally four types of devices for inhaling liposome delivery systems: pressurized metered dose inhalers (pMDIs), dry powder inhalers (DPIs), soft mist inhalers (SMIs), and medical nebulizers.¹¹⁵ pMDIs and SMIs are typically used to deliver small doses of potent drugs. In contrast, both DPIs and medical nebulizers can deliver larger drug doses. The comparison of the various devices is shown in Table 3.

Table 3.

Comparison of Pulmonary Inhalation Devices

Devices	Principle	Advantage	Disadvantage
pMDIs	A pressurized device that uses propellant sprays to deliver metered amounts of a drug solution, or suspension to the lungs.	Small size, easy to carry, active spray, low inspiratory flow rate, and highly cost-effective.	Poor coordination,^170,171 relies on propellants.¹⁷² Hydrofluoroalkanes are poor phospholipolytic agents, and few studies have chosen to use pMDIs to deliver inhaled liposomal formulations.
DPIs	A portable device that delivers the drug to the lungs as a dry powder.	Small size, easy to use, easy to carry. No propellant is needed. Minimal patient cooperation is needed for both breathing and aerosol intake.¹⁷³ Large doses can be ejected at a time.¹⁷⁴	Requirements for minimum inspiratory pressure to disperse the powder. Dry powder drugs are prone to moisture absorption, so the requirements for the packaging materials used are high.⁵⁸
SMIs	A propellant-free inhaler that utilizes mechanical power to deliver single or multiple doses of inhalable drug aerosols in the form of a slow mist to the lungs.	A longer and slower release of aerosol. Less is deposited in the mouth and throat, while relatively more is deposited in the lungs.^175–177 Requires little coordination of actuation and inhalation by patients. Currently, there is only one commercially available SMI: Respimat^® (Boehringer Ingelheim, Ingelheim am Rhein, Germany).¹⁷⁸	Few drugs are currently available for SMI.¹⁷⁸ The volume of SMIs is less than 50 μL per atomization, so it is not suitable for lung diseases (such as malignant tumors, infectious diseases) that require large doses of drugs.¹⁷⁹

DPI, dry powder inhaler; pMDI, pressurized metered dose inhaler; SMI, soft mist inhaler.

The method of preparation and deposition of liposomes in the respiratory tract depends on the types of inhalation devices. Unfortunately, the shear forces that disperse aqueous liposomal solutions into “inhalable” aerosol droplets may exert physical stress on the liposome bilayer during nebulization, resulting in loss of initially embedded drug, and jet nebulization of liquid preparations often results in excess residual fluid in the device and prolonged drug administration. Therapeutic dry powder inhalation aerosol liposome powder is formulated with phospholipids similar to the endogenous pulmonary surfactant, which provides a new method for the absorption of nanomedicine in the lung to achieve sustained release and enhanced stability.

DPI can target the treatment of a variety of lung diseases, such as lung cancer, TB, and CF. These delivery systems may only require smaller doses to be effective, with low toxicity and few side effects. The powder inhalation method can also effectively increase the stability of peptides and protein drugs and improve their bioavailability. DPIs are commonly produced via spray drying with a range of excipients, solvents, and separation options, which can be modified to improve the stability of the nucleic acid drugs.¹¹⁶ Willis et al.⁵⁸ described inhaled dry powder drugs as suitable for liposome aerosol therapy. However, there are some challenges in using DPIs with liposomes as lipid drug carriers. The main challenge is that liposomes are usually unstable in the dry state. Dehydration or conversion of liposomes to a dry state can significantly affect their effectiveness in delivering drugs, and may lose their structural characteristics and drug encapsulation properties. This limits the applicability for direct conversion into a dry powder form for inhalation.

To address this challenge, researchers are actively exploring different strategies to stabilize liposomes and maintain their properties during drying. For example, in the preparation of nanoscale liposomes (i.e., much smaller than aerosols), suitable phospholipid composition, stabilizers such as sugars or polymers are added to lipid carriers to improve stability.^117–120 These stabilizers help protect the structure of the liposomes. In addition, spray drying is the most widely used drying method for inhaled powders, and advances in spray drying technology have made it possible to develop liposome dry powders with better stability. By carefully optimizing the spray drying parameters, it is possible to prepare liposome powders that retain the key properties of liposomes such as size, stability, and drug release properties.^121–123

Given that the composition of liposomes is very similar to that of endogenous pulmonary surfactants, most liposomes are biocompatible and biodegradable. Pulmonary surfactants are a complex mixture with about 85% phospholipids, generally DPPC, followed by phosphatidylglycerol, which is thought to play an important role in the adsorption, diffusion, and reuse of surfactant.¹²⁴ The mechanisms of pulmonary surfactant clearance and reuse may be important in determining the fate of liposomes deposited in the alveoli.¹²⁵ Thus, liposome dry powder formulations can be designed to mimic endogenous pulmonary surfactant components.¹²⁶ Pulmonary surfactant has an efficient recycling mechanism, while DPPC is the major phospholipid produced and recycled by alveolar type II epithelial cells, and some lipid components can be continuously internalized and secreted by alveolar type II epithelial cells.¹²⁷

Thus, liposomes absorbed by pulmonary surfactants may be recycled in a similar manner in vivo, allowing them to remain in the alveoli for a longer time. To evaluate the effect of phospholipids in liposome formulations on pulmonary surfactant, Monforte et al.¹²⁸ treated 38 lung transplant recipients with aerosolized AmBisome (liposomal amphotericin B for injection) and showed no change in the lipid content of pulmonary surfactant, indicating that the deposition of liposomes in the lungs is safe. It does not affect the concentration of pulmonary surfactant. The 10-year follow-up study of aerosolized lung transplant recipients showed that the treatments were well tolerated, which proved that the liposomes were biocompatible in the lung tissue environment and did not cause immune reactions or toxic reactions after administration.^129,130

At present, the most common device used to simulate animal aerosol inhalation is the animal aerosol inhalation exposure device. However, these devices are usually large equipment, with complex operations, expensive, large sample requirements, large losses, and large individual variations in delivery dose, which limits their application.^131,132 An aerosol delivery system, the AERx system, has previously been reported to be capable of delivering repeatable liquid vaccine doses with properties suitable for local pulmonary therapy or systemic vaccine delivery. The device monitors inhalation velocity and provides flow rate feedback, with features needed for efficient and repeatable vaccine delivery to the lungs.^133,134

Deshpande et al. evaluated the feasibility of delivering nonviral gene therapy agents to the lungs with the AERx lung delivery system and found that “naked” DNA degraded after atomization by the AERx nozzle system. DNA prepared with a molar excess of cationic lipids (liposomes) showed no loss of integrity. In addition, a significant improvement in atomization efficiency was observed when an electrolyte such as NaCl was added to the formulation. Thus, liposome delivery technologies such as the AERx lung delivery system may be a viable approach to lung gene therapy.¹³⁵

In addition, according to the needs of vaccine aerosol immunization, Qiu et al.¹³⁶ also developed a new device for small-animal inhalation exposure to aerosols, including drugs, vaccines, microbial aerosols, PM2.5 particles, and nanoparticles, and verified the performance of the device. The experimental tests showed that the device had animal inhalation exposure and protection functions, and its performance met the requirements of relevant standards. It has been confirmed that the animal aerosol inhalation exposure device can be used for animal aerosol inhalation exposure experiments, which solves the problem of accurate and quantitative inhalation of drugs, and also guarantees the protection function of the experimental environment and personnel. It is suitable for the laboratory of toxicology, pathogenic microorganisms, vaccines, and other aerosol exposure research.

Delivery Mechanism of Liposome-Bound Nucleic Acid Vaccine

Liposome-coupled nucleic acid is a kind of amphiphilic nucleic acid compound, which can make up for the disadvantage of low immunogenicity of naked nucleic acid vaccine and enhance immune responses.^137,138 Its mechanism of action is rough as follows: (1) enhancing effectiveness and reducing toxicity,¹³⁹ (2) protective vaccine, (3) sustained release and controlled release, (4) targeting, (5) improving plasmid expression, and (6) enhanced humoral and cellular immune responses.^140,141 Promoting gene internalization into lung epithelial cells, transfection experiments in vitro showed that the atomized nanocomposites retained their gene expression function in transfected bronchial epithelial cell cultures.^142,143

Summary and Outlook

Vaccine inhalation therapy can improve the delivery of vaccines and certain active ingredients into the lungs. For this treatment, effective intrapulmonary distribution, as well as infiltration of biofilms and macrophages, has been validated. Further improvements in formulations and devices may be possible, such as enhanced liposomal mucus penetration, targeting the specificity, stability, and deposition in the lung. Liposome lung inhalation is a complex process involving multiple factors, and further research and optimization of delivery systems are needed to improve its potential for treating respiratory diseases.

At present, the question of the efficacy of the inhalation TB vaccine is still unresolved, after all, this delivery route of the vaccine still needs to overcome major biological obstacles in the lungs, such as (1) whether the cough reflex is triggered after inhalation of the vaccine and whether lung cells influence the effective uptake of the vaccine; (2) whether the liposomal formulation is degraded or rapidly cleared as it passes through mucous and epithelial barriers during delivery; (3) whether the liposomes can reach the indicated cellular or intracellular targets; (4) after reaching the target cells, the limited receptor binding or recognition and active efflux of liposomes by the cells may affect the deposition of the vaccines in alveolar macrophages. After all, macrophages have been identified as an important parasite site of Mtb.

(5) The endocytic pathway is required to overcome the cell membrane barrier so that the macromolecular vaccines cannot escape from the intracellular body and are ultimately degraded in the lysosomes; and (6) whether the liposome formulation is safe and well-tolerated. Thus, the design of liposomal formulations is critical for direct intracellular delivery.^144,145 In addition, when nanoparticles are administered by aerosol, they interact with cells, proteins, biomolecules, and physical barriers that are different from their interactions in the bloodstream, and the cells in the airway are heterogeneous. These may affect delivery and are some of the uncertainties that affect the efficacy of inhaled vaccines.^146,147

The nucleic acid vaccine molecules have poor penetration ability through the cell membrane, lack targeting, and are unstable in physiological environments. Therefore, the bottleneck of nucleic acid vaccine application in vivo lies in the construction of delivery systems for gene drugs and vaccines in vivo. Nanoparticle-conjugated vaccine technology has been successfully applied to the delivery of a variety of existing and potential vaccines. Nanoparticles with appropriate structure and size can protect the nucleic acid vaccines and enhance the immune responses in vivo.^148,149 Liposomes have become a popular nucleic acid vaccine delivery vector with great potential for development. However, there are still many problems, such as particle homogeneity and particle size in the process of liposome preparation. Liposomes with smaller particle sizes (180 nm) can significantly improve the blood concentration and bioavailability of vaccines, prolong blood circulation properties, improve bioavailability, and/or preferentially accumulate at disease sites.¹⁵⁰ Although they can increase the aggregation of active ingredients in target organs and prolong their half-life, they are prone to aggregation and fusion due to antigen leakage,^151,152 which will directly affect the specific biological effects of liposomes in vivo.¹⁵³ Cationic lipids also carry positive charges in vivo, have certain toxicity and short half-life, and may bind to other molecules with a nonspecific negative charge in the body, reducing the delivery efficiency. Therefore, it is necessary to modify or transform them to improve their delivery efficiency and stability, reduce toxicity and prolong circulation time, and target delivery to specific tissues and cells in the human body.^154,155 In addition, due to the high technical barriers, difficult quality control, lack of high-quality raw and auxiliary materials, and lack of industrialization equipment and other reasons, the progress of liposome preparation from the laboratory to industrial production is still relatively slow.

However, the silencing efficiency, off-target, and stability involved in nucleic acids remain a challenge in clinical application.¹⁵⁶ At present, lung delivery of liposome-conjugated nucleic acid vaccines may provide a more effective way to prevent and control TB. The innovation and development of lipid carriers and delivery routes will provide more therapeutic options for humans. It will also be the goal we are constantly pursuing.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Footnotes

Authors' Contributions

Conceptualization: Y.L. and X.W.; methodology: D.Z. and H.Z.; software: Y.L. and X.W.; validation: D.Z. and H.Z.; formal analysis: D.Z.; investigation: D.Z.; data curation: D.Z.; writing—original draft preparation: D.Z.; writing—review and editing: Y.L. and X.W.; visualization: D.Z. and Y.L.; supervision: Y.L.; and project administration: X.W. All the authors have read and agreed to the published version of the article.

Author Disclosure Statement

The authors declare they have no conflicting financial interests.

Funding Information

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the article; or in the decision to publish the results.

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