Optimal development of apoptotic cells-mimicking liposomes targeting macrophages | Journal of Nanobiotechnology

DoE strategy optimized the ratio of individual lipids within liposomes

The pursuit of designing efficient drug delivery systems has fueled the exploration of various formulation strategies to enhance the performance of drug carriers. Among these strategies, the DoE strategy has emerged as a powerful and systematic approach for optimizing the formulation of liposomes [25, 26]. As a versatile statistical tool, DoE enables a structured exploration of the multifaceted parameter space involved in liposomal development. In this study, pathogenic or apoptotic signals mimicking liposomes were developed and explored to achieve maximizing macrophage targeting while minimizing cytotoxicity. Mannosylated or/and phosphatidylserine (PS) -presenting liposomes were developed to mimic pathogenic and apoptotic signals. Initially, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) was selected to prepare PS-presenting liposomes, and mannose (Man) -conjugated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG) was synthesized to realize mannosylation. The synthesis of Man-PEG-DSPE was illustrated in Figure S1A, and the product was characterized by ¹H NMR. The ¹H-NMR spectrum of DSPE-PEG and Man-PEG-DSPE are shown in Figure S1B and S1C respectively. As shown in Figure S1C, the signals at 3.5–4.3 ppm represented the phenyl and hydroxyl groups in 4-aminophenyl α-D-mannopyranoside, indicating the successful reaction between the amino group and the NHS group in DSPE-PEG-NHS.

Firstly, a series of liposomes with 17 formulations (Library A) were generated by the DoE strategy by varying the molar ratio of the compositions including DPPS, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and Man-PEG-DSPE (Fig. 1A and B). The liposomes were highly uniform with particle size ranging between 80 and 100 nm, and a polydispersity index (PDI) consistently below 0.2 (Fig. 1C). As the ratio of DPPS increased, zeta potential decreased, whereas increased PEG content resulted in formulations approaching neutral zeta potentials (Fig. 1D). Cellular uptake experiments revealed a positive correlation between DPPS content and macrophage targeting efficacy, indicating the potential role of DPPS in enhancing cellular interactions (Fig. 1E and G). Cellular uptake decreased as the PEG content increased, reminding that a balanced PEG concentration to maintain targeting specificity and cellular internalization (Fig. 1E). Results from cell cytotoxicity assay showed that in most cases Chol content below 20% induced considerable toxicity, while formulations with DPPS exceeding 30%, and Chol combined with DSPC below 40%, also increased cytotoxicity. Meanwhile, DPPS content exceeding 60% was also associated with increased cytotoxicity (Fig. 1F). Based on the results above, further optimization guidelines were established, stipulating molar composition as that Chol content (20 − 50%), DSPC ≥ 20%, DPPS ≤ 60% and the molar proportion of Man-PEG-DSPE was fixed to constitute 2% of the total lipid content, which included Chol, DSPC, and DPPS. As shown in Table S1, liposomes in Library B with 9 formulations were generated and prepared. These liposomes showed a more uniform particle size (80–90 nm) as Man-PEG-DSPE fixed at 2%, and PDI was below 0.2 as well (Figure S2A). As shown in Figures S2B and S2C, zeta potential decreased and cellular uptake increased as DPPS increased, consistent with that of Library A. No obvious cell toxicity was observed when co-cultured with RAW264.7 cells for 24 h (Figure S2D). The DoE mixed formulation analysis towards cellular uptake indicated that PS modification significantly influenced the cellular uptake of liposomes by macrophages (Figure S3). Among the nine liposomal groups, those with higher levels of DPPS exhibited a notable increase in targeted cellular uptake compared to their counterparts with lower DPPS content. This underscored the pivotal role of PS in enhancing the affinity of liposomes for macrophages, promoting efficient cellular internalization. Thus, in the absence of apparent cytotoxicity, augmenting the PS content in liposomes represented a viable strategy to enhance their macrophage-targeting capabilities.

Altogether, these results highlighted the intricate interplay of liposomes’ components in influencing both targeting specificity and cytotoxicity. The use of PS modification in liposomes to mimic apoptotic signals for macrophage targeting holds significant promise for improving drug delivery precision. Results of the cellular uptake experiments have shown that the extent of PS modification in liposomes was directly correlated with the targeting efficiency towards macrophages. As the surface density of PS increases, the recognition and uptake of liposomes by macrophages also rose proportionally. This heightened targeting specificity holds promise for the precise delivery of therapeutic agents to sites associated with macrophage activation, such as inflamed tissues or tumor microenvironments. Conversely, the elevated PS modification raised concerns about the potential unwanted cytotoxic effects. The same high affinity for macrophages that enhances targeting may also result in unintended interactions with healthy cells, leading to increased toxicity. Therefore, it becomes crucial to strike a delicate balance in PS modification to achieve optimal targeting without compromising safety. As to PEG, it has been a common strategy that the incorporation of PEG into liposomes enhances their in vivo circulation time by reducing opsonization and clearance by the reticuloendothelial system. However, excessively high concentrations of PEG may compromise the efficacy of PS-modified liposomes in targeting macrophages, which was attributed to the reduced ability of PEGylated liposomes to interact with macrophages, thus hindering the specific recognition and uptake mediated by PS modification [27]. In addition, the introduction of Chol contributes to the creation of liposomes that closely mimic natural cell membranes [28]. By enhancing membrane stability, improving circulation, and modulating liposome-membrane interactions, Chol contributes to the development of biocompatible liposomal delivery systems. While Chol proves beneficial in enhancing liposome biocompatibility, the optimal Chol content in liposomal formulations requires careful consideration. Too little cholesterol may not provide the desired stabilizing effects, while excessive cholesterol can alter membrane fluidity and compromise therapeutic efficacy.

Therefore, through a thorough exploration of various lipid components via the DoE strategy, we have arrived at a refined molar ratio for the liposome formulation that optimally addresses the intricate balance between macrophage targeting and reducing cellular toxicity in liposomal formulations. The final chosen molar ratio, DSPC: DPPS: Chol: Man-PEG-DSPE = 20:60:20:2, represented a harmonious blend that caters to both macrophage targeting specificity and cellular biocompatibility.

TaE approach fine-tuned the lipid composition of liposomes

Combined with the DoE strategy, the TaE approach was employed to fine-tune liposomal formulations based on empirical observations. Iterative adjustments were made to optimize the liposomal characteristics, ensuring the replication of apoptotic cell features critical for effective macrophage targeting. This pragmatic approach complemented the systematic DoE strategy, allowing for flexibility in addressing unforeseen challenges and enhancing the likelihood of successful formulation development.

The influence of phospholipid structural variations, specifically differences in fatty acid chain length and saturation, on the targeting efficiency and toxicity of PS-presenting liposomes in macrophage interactions was a critical aspect of our study. The length of fatty acid chains in phospholipids is known to impact membrane fluidity and lipid packing. Meanwhile, the degree of saturation in fatty acid chains also plays a pivotal role in shaping liposomal characteristics. In addition, phosphatidylglycerol (PG), being a key constituent of Gram-negative bacterial cell membranes, possesses distinctive features that may influence its interactions with macrophages [29]. The negative charge associated with PG could potentially contribute to improved recognition and uptake by macrophages, mirroring the electrostatic interactions observed in bacterial-host cell interactions. This unique property of PG may open new possibilities for tailoring liposomal formulations to mimic the characteristics of Gram-negative bacteria for targeted drug delivery. Therefore, the impact of PS and PG structural modifications with varying fatty acid chain lengths and saturation levels on macrophage targeting and cytotoxicity was investigated through the TaE approach. Three distinct PS structures, namely DPPS, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), were selected for examination, alongside three PG structures—1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), and 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG)—chosen based on their comparable fatty acid chain lengths and saturation degrees (Figure S4).

Six kinds of liposomes with fixed ratios were prepared by TaE approach (Table S2). The average size was 90 nm with PDI < 0.2 (Fig. 2A). Negative potential extended with longer fatty acid chain length and higher saturation(Fig. 2B). In the context of cellular uptake experiments, the results revealed that liposomes modified with DSPS and DOPG exhibited the highest efficiency in macrophage uptake (Fig. 2C), suggesting that the specific structural features of DSPS and DOPG contribute to enhanced recognition and internalization by macrophages, pointing towards their potential as promising candidates for targeted drug delivery systems. However, it is crucial to note that, despite their high targeting efficiency, liposomes modified with DOPG demonstrated a higher level of cytotoxicity (Fig. 2D). This observation underscored the importance of carefully considering not only the targeting efficacy but also the safety profile of the modified liposomes. Balancing these factors is essential to developing effective therapeutic strategies that minimize potential adverse effects. Therefore, DSPS was chosen to replace DPPS for further study. In addition, we also evaluated three PC-structure three phospholipids with different fatty acid chain lengths and saturation of phospholipids (Figure S5). As shown in Table S3 and Fig. 2E and H, three kinds of liposomes have been made with an average size of 90 nm, PDI < 0.2 and negative potential corresponded to Fig. 2B. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) showed the highest cell uptake and no obvious cytotoxicity. Thus, DOPC was chosen to replace DSPC.

Further, we designed a series of liposomes to verify the effects of PS structure, PEG content, and Man modification on macrophage targets (Table S4). As shown in Fig. 3A and B, uniform liposomes have been prepared with an average size of 90 nm, PDI < 0.2. The lower zeta potential corresponds to increased DSPS ratio in liposomes and approached neutralization with higher PEG. In the group of 2% PEG, Man modification showed no effects on cellular uptake, whereas cell uptake increased with Man modification. Cellular uptake significantly increased in the PS group than in the PC group, however, the PEG up to 60% offset the PS effect (Fig. 3C). The most significant cellular uptake of Lipo-E6 by RAW 264.7 and THP-1 was observed by confocal laser scanning microscopy (CLSM) (Fig. 3D). Accumulated data inspired us that PS modification improves macrophage target, while Man modification did not show obvious effects on macrophage target, which could be due to the discrepancy in CD206 expression (Figure S6). Thus, PEG-PE composition of liposomes would be used for further study (final formulation DOPC: DSPS: Chol: PEG-PE = 20:60:20:2, molar ratio), and the average size was 86.1 nm and zeta potential was − 11.16 mV (Fig. 3E-H). Interestingly, the above results showed that as the PS modification increased, the zeta potential decreased, while the cellular uptake of the liposomes increased. The PS modification, despite reducing the positive charge of the liposomes, enhanced their specificity and uptake by macrophages due to the strong targeting affinity of PS for these cells. This targeted interaction is crucial for applications where precise delivery to macrophages is desired.

The observed differences in targeting efficiency among different phospholipids suggested that the structural variations in fatty acid chains contribute to the modulation of macrophage interactions. However, it is crucial to acknowledge that while certain modifications enhance targeting efficiency, they may concurrently increase cytotoxicity. Achieving an optimal balance between efficient macrophage targeting and minimal cytotoxic effects is essential for the development of safe and effective PS-modified liposomal drug delivery systems. The findings underscored the complexity of the relationship between phospholipid structure, PS/PG modification, and cellular responses. Further research and optimization efforts should aim to elucidate the underlying mechanisms governing these effects. Strategies to fine-tune the fatty acid chain characteristics, such as exploring different ratios of saturated to unsaturated lipids, may provide avenues for tailoring liposomal formulations to achieve the desired balance of targeting efficiency and reduced toxicity.

Evaluation of the potential of identified liposomes to target macrophage in murine inflammatory models

The transition from in vitro screening to in vivo validation represents a critical phase in the development of liposomal formulations for targeted therapy. Thus, following the in vitro screening process to identify the optimal liposomal formulation, three well-established murine inflammatory models, including colitis, rheumatoid arthritis (RA), and lung inflammation (Figure S7), were used to validate the targeting specificity of the optimized liposomal formulation at inflammation sites through the in vivo biodistribution imaging. As a control, liposomes composed of DSPC, Chol, and PEG-PE were used, named PC-presenting liposomes (PC-Lipo). PC-Lipo and PS-presenting liposomes (PS-Lipo) were labeled with DiR for in/ex vivo imaging.

Firstly, the in vivo distribution of PC-Lipo and PS-Lipo was investigated in healthy BALB/c mice following intravenous tail vein injection of free DiR dye, and PC-Lipo/DiR and PS-Lipo/DiR. After 24 h, the mice were euthanized, and major organs were dissected for fluorescence imaging. As shown in Figures S8 and S9, fluorescence signals of PC-Lipo/DiR group were highest in all organs, excluding the lungs. Interestingly, in the lungs, both PC-Lipo/DiR and PS-Lipo/DiR groups exhibited relatively lower fluorescence signals. The difficulty in achieving substantial lung distribution may be attributed to the intricate pulmonary microenvironment. The pulmonary environment presents unique challenges for liposomes accumulation, and the rapid blood flow and low vascular permeability in the lungs likely contribute to the reduced accumulation observed. Additionally, the observed fluorescence in other tissues indicated a potential preference for non-pulmonary organs, which may influence the overall biodistribution of these liposomal formulations. Besides, most accumulation of PC-Lipo/DiR and PS-Lipo/DiR was observed in the liver and spleen where macrophages populate [30].

Then, three murine inflammatory models were established and used to study the in vivo biodistribution of free DiR dye, PC-Lipo/DiR, and PS-Lipo/DiR. These models were selected due to the enrichment of macrophages at the sites of inflammation. In the DSS-induced colitis model (Fig. 4A), a remarkable accumulation of liposomes was observed in the intestines compared to the control group (Fig. 4C and D). Hematoxylin and eosin (H&E) staining images showed inflammation and necrosis in intestines, indicating the successful colitis model (Fig. 4B). Besides, as shown in Figure S10A and D, both immunohistochemistry (IHC) and flow cytometry experiments indicated a significant increase in macrophage infiltration in inflamed intestines. These results supported the potential of the liposomes could be recruited to the inflamed regions by macrophages. We further varied macrophage-targeting in the collagen II-induced RA model (Fig. 4E). H&E images indicated the synovium inflammation and Safranin O/Fast green staining showed the cartilage degeneration (Fig. 4F), altogether to verify the success of the RA model. Increased macrophage levels were also observed (Figure S10B and E). PS-Lipo/DiR exhibited higher fluorescence signals in the mouse paws compared to PC-Lipo/DiR (Fig. 4G and H). In addition, as shown in Figures S11 and S12, while PS-Lipo/DiR showed a higher accumulation at the sites of inflammation, its distribution in other major organs remained similar to that observed in healthy mice. These results demonstrated excellent inflammation-targeting specificity of PS-Lipo. This observation aligns with the known affinity of PS for macrophages, confirming the potential of PS modification to enhance the targeted delivery of liposomes to inflammatory sites. In a recent study, Deprez J et al. showed that the selective depletion of circulating myeloid cells reduced the accumulation of liposomes by up to 50–60%, which suggested that myeloid-cell-mediated transport accounted for more than half of the liposomal accumulation in inflamed regions [31]. This study implied that PS-presenting liposomes were likely to bind to a large number of immune cells in the bloodstream, including macrophage precursor monocytes. Theoretically, any immune cell capable of recognizing and phagocytosing PS signals has the potential to bind to PS-presenting liposomes. During inflammatory responses, an increased number of immune cells, including macrophages and their precursors, are recruited to the inflammatory sites. PS-presenting liposomes were designed to target macrophages or other PS-recognizing immune cells, enabling passive targeting of the inflammatory regions due to the increased presence of immune cells, which ensured that the liposomes were efficiently directed to the sites where they were needed most.

However, in the lipopolysaccharide (LPS)-induced lung inflammation model, a notable discrepancy was observed, and both PC-Lipo and PS-Lipo failed to reach the pulmonary tissues (Figure S13), which was consisted with the observation in healthy mice. The comparable or even lower fluorescence levels between the two groups in the lungs suggested that PS modification did not exacerbate or alleviate the challenge of lung delivery compared to PC-Lipo. The inability of PS-Lipo to reach the lungs may be attributed to the distinct pathophysiological environments of different tissues or the more widely accepted view that the formation of a protein corona affected their in vivo distribution. The formation of the protein corona significantly alters the surface characteristics of nanoparticles, thus greatly affecting their interactions with organs and cells. In a recent study, Le ND et al. synthesized a series of poly(β-amino ester) (PBAE) derivatives and found that the protein corona on nanoparticles could help achieve organ targeting (lungs and spleen) [32]. Interestingly, their findings correlated closely with the study of Min Qiu et al. [33] , which demonstrated that the protein corona on lung-targeted nanoparticles had distinct characteristics that promoted lung accumulation. The protein corona formed on the surface of liposomes is primarily influenced by the physicochemical properties of the liposomes themselves. By selecting different lipid components or varying their ratios, it is possible to achieve targeting of different organs or cells. In 2020, Daniel Siegwart et al. reported a Selective Organ Targeting (SORT) delivery technology [34]. They introduced the cationic lipid DOTAP as a fifth component to the formulation 5A2-SC8/DOPE/DMG-PEG2000/Cholesterol, thereby altering the physicochemical properties of the LNPs, including their zeta potential. This modification influenced the formation of the protein corona during blood circulation, enabling intravenous delivery of mRNA to the lungs. Recently, Daniel Siegwart’s team reporting the use of Lung SORT LNPs for delivering ABE mRNA/sgRNA to perform gene editing on lung stem cells, treating pulmonary cystic fibrosis [35]. These findings highlight the complexity and potential of manipulating nanoparticle properties and their protein corona to achieve targeted delivery to specific organs, including the lungs. Therefore, one promising avenue involves the introduction of cationic lipids into the liposomal formulation, which may introduce a more balanced surface charge to achieve lung-targeting. However, the exact mechanisms and optimal strategies require further investigation to enhance the efficacy of lung-targeted liposomal delivery systems.

In addition, pharmacokinetic experiment was conducted to investigate the pharmacokinetic profiles of PC-Lipo and PS-Lipo in mice. As shown in Fig. 5C, PS-Lipo exhibited a faster metabolic rate compared to PC-Lipo. Specifically, 50% of PS-Lipo was metabolized within the first 2 h post-injection, and nearly 80% was cleared from the body within 24 h. This rapid clearance of PS-Lipo can be attributed to the enhanced recognition and removal by the body’s clearance mechanisms, likely due to the PS modification that mimicked apoptotic signals. This facilitated the recognition and phagocytosis by immune cells, leading to quicker elimination from the body. Consequently, this rapid metabolism of PS-Lipo not only underscored its efficient clearance but also implied a potentially lower systemic exposure and toxicity, enhancing its safety profile for in vivo applications. In contrast, PC-Lipo demonstrated a slower metabolic rate, resulting in prolonged circulation time within the body. This extended presence in the bloodstream likely contributed to the notable accumulation of PC-Lipo in the liver and spleen, leading to a higher degree of uptake and accumulation in these organs compared to PS-Lipo.

Therefore, the distinct behavior of PS-Lipo in different inflammatory models underscored the context-dependent nature of their targeting efficacy. While PS modification enhanced macrophage targeting in certain inflammatory conditions, its effectiveness appeared to be compromised in the pulmonary environment. This highlighted the importance of considering the specific characteristics of each target tissue and disease context when designing targeted drug delivery systems, and potential strategies such as the introduction of cationic lipids may be explored to overcome this limitation and improve their overall efficacy in diverse inflammatory settings.

Biocompatibility evaluation in vivo

Understanding the interaction between liposomes and biological systems is essential to ensure their safety and efficacy for clinical transformation applications. Therefore, the in vivo biocompatibility of PC-Lipo and PS-Lipo has been evaluated in BALB/c mice. Mice were injected with saline, PC-Lipo, or PS-Lipo every two days, and their body weights were recorded. After two weeks, the mice were euthanized, and major tissues and blood were collected for analysis. Tissues were stained with H&E to assess for tissue damage and inflammatory reactions. As shown in Fig. 5A, no significant histological changes such as necrosis or inflammation have been observed in organs as heart, liver, spleen, lung, and kidney. Hematological parameters, encompassing red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin level (HBL), as well as biomarkers including aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CREA), collectively indicated the biocompatibility and safety of the liposomes for major organs, with no discernible toxicity observed in the hematologic system, liver, or kidney (Figure S14). The body weight was also monitored and showed a steady increase for 2 weeks (Fig. 5B), indicating that the liposomes did not induce substantial stress or alterations in the metabolic status of the animals.

These encouraging findings showed the potential application of the liposomal formulation in various therapeutic contexts with excellent biocompatibility. However, it is essential to note that this study is a preliminary assessment, and further investigations, including long-term studies and drug or bioactive molecules delivery, may contribute to providing a more comprehensive understanding of the liposomes.

Therefore, based on the current study, the prepared liposomes exhibit promising in vivo biocompatibility in female BALB/c mice. These results lay the foundation for future research and development, emphasizing the importance of continued safety assessments to ensure the translational potential of the liposomal formulation in clinical and biomedical applications.

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Tagsapoptotic cellsmimicking Development Journal liposomes macrophages Nanobiotechnology optimal targeting

Optimal development of apoptotic cells-mimicking liposomes targeting macrophages | Journal of Nanobiotechnology

DoE strategy optimized the ratio of individual lipids within liposomes

TaE approach fine-tuned the lipid composition of liposomes

Evaluation of the potential of identified liposomes to target macrophage in murine inflammatory models

Biocompatibility evaluation in vivo

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DoE strategy optimized the ratio of individual lipids within liposomes

TaE approach fine-tuned the lipid composition of liposomes

Evaluation of the potential of identified liposomes to target macrophage in murine inflammatory models

Biocompatibility evaluation in vivo

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About the author

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