Meta-Analysis of Drug Delivery Approaches for Treating Intracellular Infections

This meta-analysis aims to evaluate the trend, methodological quality and completeness of studies on intracellular delivery of antimicrobial agents. PubMed, Embase, and reference lists of related reviews were searched to identify original articles that evaluated carrier-mediated intracellular delivery and pharmacodynamics (PD) of antimicrobial therapeutics against intracellular pathogens in vitro and/or in vivo. A total of 99 studies were included in the analysis. The most commonly targeted intracellular pathogens were bacteria (62.6%), followed by viruses (16.2%) and parasites (15.2%). Twenty-one out of 99 (21.2%) studies performed neither microscopic imaging nor flow cytometric analysis to verify that the carrier particles are present in the infected cells. Only 31.3% of studies provided comparative inhibitory concentrations against a free drug control. Approximately 8% of studies, albeit claimed for intracellular delivery of antimicrobial therapeutics, did not provide any experimental data such as microscopic imaging, flow cytometry, and in vitro PD. Future research on intracellular delivery of antimicrobial agents needs to improve the methodological quality and completeness of supporting data in order to facilitate clinical translation of intracellular delivery platforms for antimicrobial therapeutics. Supplementary Information The online version contains supplementary material available at 10.1007/s11095-022-03188-z.


Introduction
Infectious disease and antimicrobial resistance remain a major threat to the public health (1). Of many causative organisms, intracellular pathogens have become a source of great concern worldwide. Intracellular pathogens have developed immune evasion strategies over evolution (2). For example, some pathogens infect macrophages, which are responsible for the eradication of invading pathogens from the host, and rather turn them to their sanctuaries to evade the host immunity (3,4). Consequently, intracellular infection caused by these organisms can often incur chronic, recurrent, or disseminated infections (5). Repeated and prolonged exposure to antimicrobials also increases the risk of turning these pathogens into multidrug resistant (MDR) or extensively drug resistant (XDR) organisms (6). Given the limited therapeutic options available and the paucity of novel antimicrobial agents in the pipeline, we face dire prospects in the global fight against intracellular infections.
To kill these pathogens, it is necessary to develop new strategies to deliver antimicrobial agents intracellularly. Ideally, antimicrobial therapeutics should selectively enter the infected cells, traffic in the cells to the desired intracellular niche (e.g., cytoplasm or vacuole) harboring pathogens, and kill the pathogens in a timely manner. The drug delivery field has a unique opportunity here, based on the knowledge obtained from the intracellular delivery of anti-cancer drugs or gene therapeutics (e.g., lipid nanoparticles of messenger ribonucleic acid (mRNA) encoding spike proteins for Coronavirus disease (COVID) vaccines (7)). In particular, gene therapeutics can contribute significantly to the therapy of intracellular infections. For example, viral infections pose further challenges to public health due to the capacity of viruses to spread and rapidly mutate rendering existing antiviral agents ineffective (7,8). Small interfering ribonucleic acids (siRNAs) are potentially useful for the development of antiviral therapy as they can be designed to adapt to genetic changes in microorganisms and address the affected populations in a timely manner (9). With the approval of mRNA COVID vaccines, we foresee accelerated development of novel products based on advanced antimicrobial agents such as siRNA for therapy of intracellular infections. Therefore, it is a prime time to review current drug carriers targeted to intracellular pathogens and understand the achievements, limitations, and opportunities.
A wide variety of carrier systems have been investigated in the literature, ranging from nanoparticles, microparticles, live cell derivatives, prodrugs, or drug conjugates. Many studies employ particles as a carrier of antimicrobial agents, exploiting the ability of host cells (mostly macrophages) to endocytose them. The particles are made of organic or inorganic compounds that can encapsulate antimicrobial agents, in a size conducive to cellular uptake. Since mammalian cells take up particles via dedicated endocytosis pathways, the encapsulated drugs may bypass diffusional barrier imposed by the cell membrane (10) (Fig. 1). Ninety-nine papers on this topic were published in the past decade, with 89.9% of them in the last 10 years. However, there are only a limited number of commercial products based on the intracellular drug delivery approach, with silver nanoparticles being the most widely studied nanoparticles (11).
In this study, we aim to better understand the current landscape of drug delivery systems targeted to intracellular pathogens and potential barriers in their clinical translation. We performed a meta-analysis of literature concerning drug-loaded platforms targeting intracellular pathogens and reviewed study methodologies for evaluating their intracellular delivery and antimicrobial efficacy. Previous metaanalyses on drug-loaded platforms have primarily focused on anticancer therapy (12,13). To the best of our knowledge, the present study is the first meta-analysis on carrier-mediated delivery of antimicrobial therapeutics against intracellular pathogens.

Data Source and Search Strategy
A literature search of PubMed and Embase was performed to identify original articles that evaluated carrier-mediated intracellular delivery and/or antimicrobial efficacy of antimicrobial therapeutics against intracellular pathogens in vitro or in vivo. Here, antimicrobial therapeutics are defined broadly to include antiviral, antibacterial, antifungal, and antiparasitic agents. The initial database search strategies were prespecified as follows: search terms consisted of a combination of key words, (intracellular) in title/abstract AND (infection) in title/abstract AND (delivery) in title/ abstract; no publication date restrictions were applied; the search filter for language was set as "English." Reference lists of related research were additionally screened to identify eligible studies. Ethics committee review was waived as this study conducted by pooling existing data extracted from published primary research.

Study Selection and Data Collection
Those studies identified though initial search were screened for eligibility using the following inclusion criteria: (i) experimental data are provided to demonstrate the efficacy of intracellular delivery of antimicrobial therapeutics and/or their antimicrobial efficacy against intracellular pathogens in vitro or in vivo; (ii) carrier systems are used to deliver active drugs; (iii) sufficient raw data can be obtained from primary research. Review articles and duplicate studies were excluded from the meta-analysis. Studies that applied metals or microorganisms for preparation of intracellular delivery platforms were included. Study attributes, including the first author and year of publication, host cells, etiologic organisms, characteristics of carrier, types of materials (organic, inorganic, live cell derivative), active drug, intracellular delivery study methodology, in vitro and/or in vivo pharmacodynamic (PD) markers, antimicrobial efficacy results, and free drug control were extracted utilizing a predefined summary format. Authors resolved difference in opinion or study interpretation through discussion.

Data Analysis and Statistical Methods
The efficacy of intracellular carriers for antimicrobial therapeutics was evaluated qualitatively and quantitatively by pooling data from individual studies, and the results were presented with descriptive statistics. The parameters of intracellular delivery investigation assessed in this meta-analysis included what type of study methodologies were used for evaluation of intracellular delivery (microscopy, flow cytometry), whether in vitro and/or in vivo PD responses were evaluated, and whether the effects were compared against those of free drug counterparts. The primary in vitro PD markers investigated are: intracellular microbial burdens such as colony forming units (CFUs), viral plagues or titers, optical density, and microbial growth or inhibition rates; inhibitory concentrations including minimum inhibitory concentration (MIC), half maximal inhibitory concentration (IC 50 ), and half maximal effective concentration (EC 50 ). As for in vivo PD markers, survival rates and pathogen burden at specific times were assessed in animal models of related infection.

Selection of Relevant Original Studies
The process of searching and identifying relevant studies is presented as Fig. 2. The initial search from PubMed and Embase yielded 1,891 citations and 67 additional articles were identified via reference lists in reviews of related research (14)(15)(16)(17). Of those, 700 duplicates were excluded. Of the remaining 1,258 articles that underwent further screening of titles and abstracts, a total of 1,131 articles were excluded due to the reasons listed in Fig. 2

Characteristics of Included Studies
The yearly publication patterns of intracellular delivery of antimicrobial therapeutics show near-zero publications until the late 2000s and a sharp uptake in publication volume in the 2010s and thereafter (Fig. 3). The most commonly targeted intracellular pathogens were bacterial organisms throughout the study screening period, with the first study on Salmonella published in 1990 followed by scarce research until a sharp rise in publication volume in the late 2000s. None of the studies targeted parasitic, viral, or fungal intracellular pathogens until 2009, 2010, and 2015, respectively, with the first studied organism being Leishmania, Ebola, and Cryptococcus, respectively. The most dominant type of materials for delivery platforms was organic compounds, of which 54.5% of studies (54/99) employed synthetic polymers and 33.3% (33/99) lipid particles (Fig. 4a). The most-commonly used carrier type was nanoparticles (in 51 out of 99 studies), followed by liposomes (in 15 out of 99 studies) (Fig. 4b). The particle size in the majority of studies (63.6%) falls in the range of 100 to < 1000 nm (Fig. 4c). We have further analyzed the included studies by host cell types, carrier types, particle size ranges, and administration routes. Detailed results are summarized in Tables S1 to S8 in supplementary materials.

Bacterial Intracellular Pathogens
The 62 eligible studies on bacterial intracellular pathogens are summarized in Table I. The most commonly studied intracellular bacteria were Staphylococcus aureus (n = 20), followed by Mycobacterium tuberculosis (n = 17) and Salmonella (n = 9) (Fig. 5). There were two studies by Lueth (2019) (18) and by Omolo (2021) (19), which provided the most comprehensive data supporting intracellular delivery and antimicrobial efficacy of drug-loaded carriers both in   In the former, a polymer-based nanoparticle was developed to encapsulate doxycycline and rifampicin to eradicate Brucella melitensis or Escherichia coli (18). Intracellular delivery was confirmed by microscopic imaging. In the latter, pH-responsive liposomes were produced to load vancomycin to kill methicillin-resistant Staphylococcus aureus (MRSA) (19). Here, intracellular delivery was confirmed by flow cytometry. In both studies, antibacterial activity (changes in MIC and CFU of intracellular bacteria) was compared against those of a free drug control in vitro, where the encapsulated drug showed more efficient bacterial killing than the free drug counterpart. The in vitro results were further validated in mouse models of systemic or topical bacterial infection (CFU changes). The distribution of particles in the main organs of interest (liver and spleen) were determined only in the former study.

Viral Intracellular Pathogens
Table II summarizes the 16 intracellular delivery studies of antimicrobial therapeutics targeting viral pathogens. The most commonly studied intracellular viruses were human immunodeficiency virus (n = 7), followed by hepatitis C virus (n = 3) (Fig. 5). A 2018 study by Hu et al. (80) showed the most comprehensive data for intracellular delivery of diphyllin or bafilomycin and their antimicrobial efficacy. The drug was encapsulated in nanoparticles prepared with    50 and viral titers were compared with those of free drug controls. In an in vivo mouse model of influenza infection, survival rates and viral RNA copies were measured as PD markers. The delivery of intravenously injected particles to the lungs, the organ of interest, was demonstrated based on the reduction of viral loads, but nanoparticle distribution in other organs was not examined.

Fungal Intracellular Pathogens
The 8 studies on fungal intracellular infection are summarized in Table III. The most commonly studied intracellular fungi were Candida albicans (n = 4) (Fig. 5). In an exemplary study by Batista-Duharte (96), a needle-like lipid assembly was prepared to deliver amphotericin B intracellularly to kill Sporothrix schenckii. Microscopic imaging data were provided to confirm intracellular delivery. The in vitro antifungal activity, in terms of CFU and MIC changes, of the carrier-mediated delivery was superior to that of a free drug. The antifungal effects were validated in a mouse model of systemic fungal infection with CFUs as a PD marker. The liver and spleen of the treated animals were examined to evaluate the fungal load.  (Fig. 5). Three studies (104)(105)(106) incorporated the most comprehensive supporting data. Intracellular delivery of particles was confirmed by microscopy and flow cytometry in all three studies. In vitro killing effects (parasitic burden and IC 50 changes) were compared with those of free drug controls. All three studies demonstrated enhanced in vivo parasite inhibition effects of the carrier-mediated delivery versus a free drug. The biodistribution of intravenously injected carrier particles was investigated in two studies, based on drugs delivered to major organs, showing that the liver and spleen were common targets (104,105).

Study Methodologies: Intracellular Delivery and Antimicrobial Efficacy
We investigated how intracellular drug delivery was evaluated in each study: imaging and flow cytometric analysis of labeled carriers, in vitro PD (e.g., killing of intracellular bacteria or counting viral plaques), or in vivo PD. Table V summarizes study methodologies employed in the analyzed

Discussion
Intracellular pathogens have evolved to counterbalance the host immunity to survive or multiplicate within macrophages, otherwise hostile environments. In order to treat intracellular infections, it is important to develop an effective intracellular delivery system that helps antimicrobial therapeutics to enter the infected cells, traffic in the cells to the desired intracellular niche harboring pathogens, and kill the pathogens in a timely manner. In this meta-analysis, we performed a descriptive and qualitative evaluation of the studies focused on carrier development for the intracellular delivery of antimicrobial agents. Unlike drug delivery studies in the cancer arena, most intracellular pathogens-targeting studies do not report intracellular drug concentrations, percentage injected dose (%ID) or mg/kg from infected cells or target organs following drug administration. Therefore, not enough raw data were available to perform physiological and pharmacokinetic model-based analyses. Of all the bacterial, viral, fungal, and parasitic intracellular pathogens, bacteria were the most common microbes studied, with Staphylococcus aureus and Mycobacterium tuberculosis being the top two etiologic organisms. They are well known for their ability to acquire antibiotic resistance, and one of the mechanisms behind the resistance is to parasitize macrophages. Notably, Leishmania were the sole species that were chosen as a target pathogen in the parasite category. Leishmaniasis is one of the deadliest infections caused by the obligate intracellular protozoa, by which more than 1.5 million people are newly affected and about 70,000 deaths occur each year (117). It can be transmitted via sand flies, and about 70 animals including humans are known hosts of Leishmania (118). There are other protozoan parasites, such as Toxoplasma gondii and Trypanosoma cruzi, which take advantage of phagocytes to grow, replicate, and evade the host immune system. However, Leishmania species differ from other protozoan parasites in that they predominantly exploit macrophages as host cells (119). Pentavalent antimonial compounds, for example meglumine antimoniate, are the mainstay of treatment but with severe side effects, such as hepatotoxicity and cardiotoxicity (120). Due to the severity of the disease and many challenges that it poses against mammalian populations, there have been a steady and increasing number of studies targeting Leishmania.
Our analysis shows that seventy-four (74.7%) out of 99 studies are done with macrophages (Table S1). The predominance of macrophages as the host cells is attributed to their role in innate immunity as the first defense to the invading bacteria (121). The most common administration route used in in vivo PD studies was intravenous injection (in 25 (58.1%) out of 43 studies) (Table S5). Upon administration, drug-loaded nanocarriers undergo opsonization, distribution in the reticuloendothelial system (RES) organs, and subsequent phagocytic uptake predominantly by macrophages. The particle size in the majority of studies (63.6%) falls in the range of 100 to < 1000 nm (Fig. 4c, Table S3), which is likely driven by the administration route (intravenous injection) and biodistribution in the RES organs where the most infected phagocytes are located (14,35). The most commonly used carrier type was nanoparticles followed by liposomes, in 51 (51.5%) and 15 (15.2%) out of 99 studies, respectively (Fig. 4b, Table S7), some of which contained mannose or β-glucan ligands to target receptors (e.g., CD206 or Dectin 1) expressed on macrophages.
Most studies (78.8%) provided some proof of intracellular delivery based on microscopic imaging of labeled carriers in the cells or flow cytometric quantitation of cellular fluorescence after incubation with the carriers, but not to the organelle levels: how the carriers traffic in the cells and escape endosomes to finally reach the intracellular niche where the pathogens are hiding. We found that only 31 out of 99 studies provided comparative inhibitory concentrations (MIC, IC 50 , or EC 50 ) against a free drug control. There were 8 studies that claimed their carrier system was for intracellular delivery of antimicrobial therapeutics but did not provide any experimental evidence supporting such claims, such as microscopic imaging, flow cytometry, and in vitro PD.
While microscopy and flow cytometry are dominant tools to investigate intracellular delivery of carriers, the following limitations need to be considered in interpreting the results. To enable microscopic or flow cytometric detection of carriers, fluorescent dyes are incorporated in the carriers by chemical conjugation or physical encapsulation (122). In the former, the fluorescent signals indicate the carriers (provided that the conjugation is stable throughout the experiment). In the latter, the fluorescent dye is considered a proxy of the delivered drug. Both cases have caveats. It is often assumed that the intracellular delivery of a carrier is synonymous to intracellular delivery of a drug. It is not always the case if the drug is prematurely released before the carrier enters the cells. Drug release profile measured in buffered saline does not warrant stable drug encapsulation since serum proteins can accelerate the drug release (123); therefore, one may not exclude the possibility of premature drug release in vivo on the basis of in vitro release kinetics. On the other hand, when a dye is physically encapsulated in the delivery platform, intracellular fluorescent signal only indicates that the dye can be delivered or released into the cell. While this result may provide a reasonable anticipation that a drug may be able to enter the cells likewise, it cannot be taken for granted since the dye does not always represent physical and chemical properties of the therapeutic agents. Moreover, some of the lipophilic dyes may leach out of carriers and partition to the cell membrane upon the carrier-to-membrane contact, without the carrier entering the cells (122). In this case, the fluorescence-based methods can produce a misleading conclusion about the carrier. Therefore, the results from both methods may not be used as sole evidence for intracellular delivery of drugs. If these methods indicate intracellular delivery of carriers and drug surrogates, additional studies need to be performed with drug-loaded carriers to confirm the intracellular delivery of a drug. For example, drug concentration can be measured in vitro after treating the cells with the drug-loaded carriers. Nevertheless, the entry of drug into cells via the designed carrier does not warrant intracellular trafficking to the target organelles such as endoplasmic reticulum-like vacuoles, late endosomal compartments, and macrophage phagosomes. Therefore, it is preferable to complement with in vitro PD tests, such as intracellular CFU.
To understand the contribution of carriers to intracellular delivery of a drug, control groups need to be selected carefully. When we surveyed the published reports, 69.7% have compared the in vitro antimicrobial efficacy of encapsulated therapeutic agents to the free control. Of these, only 31.3% provided comparative MIC, IC 50 , or EC 50 versus that of the free drug control. Given the potential bioactivity of carriers, it is desirable to include a drug-free carrier and a mixture of drug and drug-free carrier as additional controls. For example, chitosan or fucoidan, one of the commonly used carrier materials, have shown to induce antibacterial activity (124,125). Without a comparison with an unformulated drug, drug-free carrier, and their mixture, it is difficult to determine whether the enhanced therapeutic efficacy of a carrierloaded drug is due to the encapsulation and intracellular  delivery of the drug or to the additive/synergistic activities of the drug and carrier. Although the blank delivery platform control was not employed as a quality evaluation criterion in this meta-analysis, it would be ideal to include not only free drugs but also drug-free carriers when comparing the PD in future studies. Our analysis also reveals an access barrier that formulation scientists may face in entering the antimicrobial delivery field. Many organisms of clinical significance require a laboratory with the biosafety level 3 or higher and biocontainment facilities due to the infectious nature of microorganisms (Mycobacterium tuberculosis, Francisella tularensis, Brucella, Ebola, etc.) (126). Non-pathogenic models that share the intracellular residence and drug sensitivity as pathogens of interest will be extremely valuable to drug delivery scientists who intend to assess the effectiveness of delivery systems. For instance, Mycobacterium smegmatis was once considered as a non-pathogenic, fast-growing surrogate of M. tuberculosis (127,128). Although M. smegmatis was criticized for the differential sensitivity to drugs and stresses and the inability to persist in phagocytes (129), a new strain similar to M. tuberculosis in drug sensitivity has been reported in recent literature (130). We expect that growing availability of such non-pathogenic model organisms will help introduce more investigators into the antimicrobial drug delivery field. Animal models are another bottleneck to the translation of new technology. M. tuberculosis has been studied in various preclinical models, including zebrafish, mice, rabbits, guinea pigs, and nonhuman primates, infected with model organisms (37,(131)(132)(133)(134)(135)(136). The sensitivity to infection, immune responses to the pathogens, and the type of lesions vary with animal models; thus, the choice of animal models depends on the purpose of research (131). In the evaluation of new carriers, mouse models are dominantly used for locating the carriers in organ-and cell levels. However, a recent study shows an example using zebrafish to observe in vivo trafficking of polymersomes to M. marinum-infected macrophages and granulomas with the convenience of optical transparency and fluorescent transgenic lines (37). To determine the effectiveness of new delivery technology in cost-and time-efficient manner, it will be important for drug delivery scientists to develop broader understanding of available animal models and their utility and limitations.

Conclusions
Substantial advancements have been made in nanotechnology and precision medicine over the last few decades. The present meta-analysis reviewed the quality of carriermedicated antimicrobial delivery studies against intracellular pathogens and revealed that several studies lack essential components of PD efficacy studies. To facilitate the clinical translation of antimicrobial products for intracellular targets, future drug delivery research on intracellular pathogens need to consider the utility and limitations of dominant experimental methodologies and improve the rigor of carrier evaluation.