Optimum improvement of apoptotic cells-mimicking liposomes concentrating on macrophages | Journal of Nanobiotechnology

DoE technique optimized the ratio of particular person lipids inside liposomes

The pursuit of designing environment friendly drug supply programs has fueled the exploration of varied formulation methods to reinforce the efficiency of drug carriers. Amongst these methods, the DoE technique has emerged as a robust and systematic strategy for optimizing the formulation of liposomes [25, 26]. As a flexible statistical instrument, DoE allows a structured exploration of the multifaceted parameter house concerned in liposomal improvement. On this examine, pathogenic or apoptotic alerts mimicking liposomes have been developed and explored to realize maximizing macrophage concentrating on whereas minimizing cytotoxicity. Mannosylated or/and phosphatidylserine (PS) -presenting liposomes have been developed to imitate pathogenic and apoptotic alerts. Initially, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) was chosen to arrange 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]. As well as, the introduction of Chol contributes to the creation of liposomes that carefully mimic pure cell membranes [28]. By enhancing membrane stability, bettering circulation, and modulating liposome-membrane interactions, Chol contributes to the event of biocompatible liposomal supply programs. Whereas Chol proves helpful in enhancing liposome biocompatibility, the optimum Chol content material in liposomal formulations requires cautious consideration. Too little ldl cholesterol might not present the specified stabilizing results, whereas extreme ldl cholesterol can alter membrane fluidity and compromise therapeutic efficacy.

Subsequently, via an intensive exploration of varied lipid parts through the DoE technique, we’ve got arrived at a refined molar ratio for the liposome formulation that optimally addresses the intricate steadiness between macrophage concentrating on and lowering mobile toxicity in liposomal formulations. The ultimate chosen molar ratio, DSPC: DPPS: Chol: Man-PEG-DSPE = 20:60:20:2, represented a harmonious mix that caters to each macrophage concentrating on specificity and mobile biocompatibility.

TaE strategy fine-tuned the lipid composition of liposomes

Mixed with the DoE technique, the TaE strategy was employed to fine-tune liposomal formulations primarily based on empirical observations. Iterative changes have been made to optimize the liposomal traits, making certain the replication of apoptotic cell options important for efficient macrophage concentrating on. This pragmatic strategy complemented the systematic DoE technique, permitting for flexibility in addressing unexpected challenges and enhancing the probability of profitable formulation improvement.

The affect of phospholipid structural variations, particularly variations in fatty acid chain size and saturation, on the concentrating on effectivity and toxicity of PS-presenting liposomes in macrophage interactions was a important facet of our examine. The size of fatty acid chains in phospholipids is thought to affect membrane fluidity and lipid packing. In the meantime, the diploma of saturation in fatty acid chains additionally performs a pivotal function in shaping liposomal traits. As well as, phosphatidylglycerol (PG), being a key constituent of Gram-negative bacterial cell membranes, possesses distinctive options that will affect its interactions with macrophages [29]. The detrimental cost related to PG might probably contribute to improved recognition and uptake by macrophages, mirroring the electrostatic interactions noticed in bacterial-host cell interactions. This distinctive property of PG might open new potentialities for tailoring liposomal formulations to imitate the traits of Gram-negative micro organism for focused drug supply. Subsequently, the affect of PS and PG structural modifications with various fatty acid chain lengths and saturation ranges on macrophage concentrating on and cytotoxicity was investigated via the TaE strategy. Three distinct PS constructions, particularly DPPS, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), have been chosen for examination, alongside three PG constructions—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 primarily based on their comparable fatty acid chain lengths and saturation levels (Determine S4).

Six sorts of liposomes with mounted ratios have been ready by TaE strategy (Desk S2). The typical dimension was 90 nm with PDI < 0.2 (Fig. 2A). Detrimental potential prolonged with longer fatty acid chain size and better saturation(Fig. 2B). Within the context of mobile uptake experiments, the outcomes revealed that liposomes modified with DSPS and DOPG exhibited the very best effectivity in macrophage uptake (Fig. 2C), suggesting that the precise structural options of DSPS and DOPG contribute to enhanced recognition and internalization by macrophages, pointing in direction of their potential as promising candidates for focused drug supply programs. Nevertheless, it’s essential to notice that, regardless of their excessive concentrating on effectivity, liposomes modified with DOPG demonstrated the next stage of cytotoxicity (Fig. 2D). This remark underscored the significance of fastidiously contemplating not solely the concentrating on efficacy but additionally the protection profile of the modified liposomes. Balancing these components is important to growing efficient therapeutic methods that reduce potential opposed results. Subsequently, DSPS was chosen to interchange DPPS for additional examine. As well as, we additionally evaluated three PC-structure three phospholipids with completely different fatty acid chain lengths and saturation of phospholipids (Determine S5). As proven in Desk S3 and Fig. 2E and H, three sorts of liposomes have been made with a median dimension of 90 nm, PDI < 0.2 and detrimental potential corresponded to Fig. 2B. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) confirmed the very best cell uptake and no apparent cytotoxicity. Thus, DOPC was chosen to interchange DSPC.

Additional, we designed a sequence of liposomes to confirm the consequences of PS construction, PEG content material, and Man modification on macrophage targets (Desk S4). As proven in Fig. 3A and B, uniform liposomes have been ready with a median dimension of 90 nm, PDI < 0.2. The decrease zeta potential corresponds to elevated DSPS ratio in liposomes and approached neutralization with increased PEG. Within the group of two% PEG, Man modification confirmed no results on mobile uptake, whereas cell uptake elevated with Man modification. Mobile uptake considerably elevated within the PS group than within the PC group, nonetheless, the PEG as much as 60% offset the PS impact (Fig. 3C). Probably the most important mobile uptake of Lipo-E6 by RAW 264.7 and THP-1 was noticed by confocal laser scanning microscopy (CLSM) (Fig. 3D). Collected knowledge impressed us that PS modification improves macrophage goal, whereas Man modification didn’t present apparent results on macrophage goal, which may very well be as a result of discrepancy in CD206 expression (Determine S6). Thus, PEG-PE composition of liposomes could be used for additional examine (remaining formulation DOPC: DSPS: Chol: PEG-PE = 20:60:20:2, molar ratio), and the typical dimension was 86.1 nm and zeta potential was − 11.16 mV (Fig. 3E-H). Apparently, the above outcomes confirmed that because the PS modification elevated, the zeta potential decreased, whereas the mobile uptake of the liposomes elevated. The PS modification, regardless of lowering the constructive cost of the liposomes, enhanced their specificity and uptake by macrophages as a result of sturdy concentrating on affinity of PS for these cells. This focused interplay is essential for functions the place exact supply to macrophages is desired.

The noticed variations in concentrating on effectivity amongst completely different phospholipids advised that the structural variations in fatty acid chains contribute to the modulation of macrophage interactions. Nevertheless, it’s essential to acknowledge that whereas sure modifications improve concentrating on effectivity, they could concurrently enhance cytotoxicity. Attaining an optimum steadiness between environment friendly macrophage concentrating on and minimal cytotoxic results is important for the event of secure and efficient PS-modified liposomal drug supply programs. The findings underscored the complexity of the connection between phospholipid construction, PS/PG modification, and mobile responses. Additional analysis and optimization efforts ought to intention to elucidate the underlying mechanisms governing these results. Methods to fine-tune the fatty acid chain traits, comparable to exploring completely different ratios of saturated to unsaturated lipids, might present avenues for tailoring liposomal formulations to realize the specified steadiness of concentrating on effectivity and lowered toxicity.

Analysis of the potential of recognized liposomes to focus on macrophage in murine inflammatory fashions

The transition from in vitro screening to in vivo validation represents a important section within the improvement of liposomal formulations for focused remedy. Thus, following the in vitro screening course of to establish the optimum liposomal formulation, three well-established murine inflammatory fashions, together with colitis, rheumatoid arthritis (RA), and lung irritation (Determine S7), have been used to validate the concentrating on specificity of the optimized liposomal formulation at irritation websites via the in vivo biodistribution imaging. As a management, liposomes composed of DSPC, Chol, and PEG-PE have been used, named PC-presenting liposomes (PC-Lipo). PC-Lipo and PS-presenting liposomes (PS-Lipo) have been labeled with DiR for in/ex vivo imaging.

Firstly, the in vivo distribution of PC-Lipo and PS-Lipo was investigated in wholesome 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 have been euthanized, and main organs have been dissected for fluorescence imaging. As proven in Figures S8 and S9, fluorescence alerts of PC-Lipo/DiR group have been highest in all organs, excluding the lungs. Apparently, within the lungs, each PC-Lipo/DiR and PS-Lipo/DiR teams exhibited comparatively decrease fluorescence alerts. The issue in attaining substantial lung distribution could also be attributed to the intricate pulmonary microenvironment. The pulmonary setting presents distinctive challenges for liposomes accumulation, and the speedy blood stream and low vascular permeability within the lungs possible contribute to the lowered accumulation noticed. Moreover, the noticed fluorescence in different tissues indicated a possible desire for non-pulmonary organs, which can affect the general biodistribution of those liposomal formulations. In addition to, most accumulation of PC-Lipo/DiR and PS-Lipo/DiR was noticed within the liver and spleen the place macrophages populate [30].

Then, three murine inflammatory fashions have been established and used to check the in vivo biodistribution of free DiR dye, PC-Lipo/DiR, and PS-Lipo/DiR. These fashions have been chosen as a result of enrichment of macrophages on the websites of irritation. Within the DSS-induced colitis mannequin (Fig. 4A), a outstanding accumulation of liposomes was noticed within the intestines in comparison with the management group (Fig. 4C and D). Hematoxylin and eosin (H&E) staining pictures confirmed irritation and necrosis in intestines, indicating the profitable colitis mannequin (Fig. 4B). In addition to, as proven in Determine S10A and D, each immunohistochemistry (IHC) and stream cytometry experiments indicated a big enhance in macrophage infiltration in infected intestines. These outcomes supported the potential of the liposomes may very well be recruited to the infected areas by macrophages. We additional various macrophage-targeting within the collagen II-induced RA mannequin (Fig. 4E). H&E pictures indicated the synovium irritation and Safranin O/Quick inexperienced staining confirmed the cartilage degeneration (Fig. 4F), altogether to confirm the success of the RA mannequin. Elevated macrophage ranges have been additionally noticed (Determine S10B and E). PS-Lipo/DiR exhibited increased fluorescence alerts within the mouse paws in comparison with PC-Lipo/DiR (Fig. 4G and H). As well as, as proven in Figures S11 and S12, whereas PS-Lipo/DiR confirmed the next accumulation on the websites of irritation, its distribution in different main organs remained just like that noticed in wholesome mice. These outcomes demonstrated wonderful inflammation-targeting specificity of PS-Lipo. This remark aligns with the identified affinity of PS for macrophages, confirming the potential of PS modification to reinforce the focused supply of liposomes to inflammatory websites. In a current examine, Deprez J et al. confirmed that the selective depletion of circulating myeloid cells lowered the buildup of liposomes by as much as 50–60%, which advised that myeloid-cell-mediated transport accounted for greater than half of the liposomal accumulation in infected areas [31]. This examine implied that PS-presenting liposomes have been prone to bind to a lot of immune cells within the bloodstream, together with macrophage precursor monocytes. Theoretically, any immune cell able to recognizing and phagocytosing PS alerts has the potential to bind to PS-presenting liposomes. Throughout inflammatory responses, an elevated variety of immune cells, together with macrophages and their precursors, are recruited to the inflammatory websites. PS-presenting liposomes have been designed to focus on macrophages or different PS-recognizing immune cells, enabling passive concentrating on of the inflammatory areas as a result of elevated presence of immune cells, which ensured that the liposomes have been effectively directed to the websites the place they have been wanted most.

Nevertheless, within the lipopolysaccharide (LPS)-induced lung irritation mannequin, a notable discrepancy was noticed, and each PC-Lipo and PS-Lipo failed to achieve the pulmonary tissues (Determine S13), which was consisted with the remark in wholesome mice. The comparable and even decrease fluorescence ranges between the 2 teams within the lungs advised that PS modification didn’t exacerbate or alleviate the problem of lung supply in comparison with PC-Lipo. The lack of PS-Lipo to achieve the lungs could also be attributed to the distinct pathophysiological environments of various tissues or the extra extensively accepted view that the formation of a protein corona affected their in vivo distribution. The formation of the protein corona considerably alters the floor traits of nanoparticles, thus tremendously affecting their interactions with organs and cells. In a current examine, Le ND et al. synthesized a sequence of poly(β-amino ester) (PBAE) derivatives and located that the protein corona on nanoparticles might assist obtain organ concentrating on (lungs and spleen) [32]. Apparently, their findings correlated carefully with the examine of Min Qiu et al. [33] , which demonstrated that the protein corona on lung-targeted nanoparticles had distinct traits that promoted lung accumulation. The protein corona shaped on the floor of liposomes is primarily influenced by the physicochemical properties of the liposomes themselves. By choosing completely different lipid parts or various their ratios, it’s attainable to realize concentrating on of various organs or cells. In 2020, Daniel Siegwart et al. reported a Selective Organ Concentrating on (SORT) supply expertise [34]. They launched the cationic lipid DOTAP as a fifth element to the formulation 5A2-SC8/DOPE/DMG-PEG2000/Ldl cholesterol, thereby altering the physicochemical properties of the LNPs, together with their zeta potential. This modification influenced the formation of the protein corona throughout blood circulation, enabling intravenous supply of mRNA to the lungs. Not too long ago, Daniel Siegwart’s group reporting using Lung SORT LNPs for delivering ABE mRNA/sgRNA to carry out gene modifying on lung stem cells, treating pulmonary cystic fibrosis [35]. These findings spotlight the complexity and potential of manipulating nanoparticle properties and their protein corona to realize focused supply to particular organs, together with the lungs. Subsequently, one promising avenue entails the introduction of cationic lipids into the liposomal formulation, which can introduce a extra balanced floor cost to realize lung-targeting. Nevertheless, the precise mechanisms and optimum methods require additional investigation to reinforce the efficacy of lung-targeted liposomal supply programs.

As well as, pharmacokinetic experiment was performed to research the pharmacokinetic profiles of PC-Lipo and PS-Lipo in mice. As proven in Fig. 5C, PS-Lipo exhibited a sooner metabolic fee in comparison with PC-Lipo. Particularly, 50% of PS-Lipo was metabolized throughout the first 2 h post-injection, and almost 80% was cleared from the physique inside 24 h. This speedy clearance of PS-Lipo might be attributed to the improved recognition and removing by the physique’s clearance mechanisms, possible as a result of PS modification that mimicked apoptotic alerts. This facilitated the popularity and phagocytosis by immune cells, resulting in faster elimination from the physique. Consequently, this speedy metabolism of PS-Lipo not solely underscored its environment friendly clearance but additionally implied a probably decrease systemic publicity and toxicity, enhancing its security profile for in vivo functions. In distinction, PC-Lipo demonstrated a slower metabolic fee, leading to extended circulation time throughout the physique. This prolonged presence within the bloodstream possible contributed to the notable accumulation of PC-Lipo within the liver and spleen, resulting in the next diploma of uptake and accumulation in these organs in comparison with PS-Lipo.

Subsequently, the distinct habits of PS-Lipo in numerous inflammatory fashions underscored the context-dependent nature of their concentrating on efficacy. Whereas PS modification enhanced macrophage concentrating on in sure inflammatory circumstances, its effectiveness gave the impression to be compromised within the pulmonary setting. This highlighted the significance of contemplating the precise traits of every goal tissue and illness context when designing focused drug supply programs, and potential methods such because the introduction of cationic lipids could also be explored to beat this limitation and enhance their total efficacy in numerous inflammatory settings.

Biocompatibility analysis in vivo

Understanding the interplay between liposomes and organic programs is important to make sure their security and efficacy for scientific transformation functions. Subsequently, the in vivo biocompatibility of PC-Lipo and PS-Lipo has been evaluated in BALB/c mice. Mice have been injected with saline, PC-Lipo, or PS-Lipo each two days, and their physique weights have been recorded. After two weeks, the mice have been euthanized, and main tissues and blood have been collected for evaluation. Tissues have been stained with H&E to evaluate for tissue injury and inflammatory reactions. As proven in Fig. 5A, no important histological adjustments comparable to necrosis or irritation have been noticed in organs as coronary heart, liver, spleen, lung, and kidney. Hematological parameters, encompassing purple blood cell (RBC) depend, white blood cell (WBC) depend, hemoglobin stage (HBL), in addition to biomarkers together with aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CREA), collectively indicated the biocompatibility and security of the liposomes for main organs, with no discernible toxicity noticed within the hematologic system, liver, or kidney (Determine S14). The physique weight was additionally monitored and confirmed a gentle enhance for two weeks (Fig. 5B), indicating that the liposomes didn’t induce substantial stress or alterations within the metabolic standing of the animals.

These encouraging findings confirmed the potential utility of the liposomal formulation in varied therapeutic contexts with wonderful biocompatibility. Nevertheless, it’s important to notice that this examine is a preliminary evaluation, and additional investigations, together with long-term research and drug or bioactive molecules supply, might contribute to offering a extra complete understanding of the liposomes.

Subsequently, primarily based on the present examine, the ready liposomes exhibit promising in vivo biocompatibility in feminine BALB/c mice. These outcomes lay the muse for future analysis and improvement, emphasizing the significance of continued security assessments to make sure the translational potential of the liposomal formulation in scientific and biomedical functions.

DoE technique optimized the ratio of particular person lipids inside liposomes

TaE strategy fine-tuned the lipid composition of liposomes

Analysis of the potential of recognized liposomes to focus on macrophage in murine inflammatory fashions

Biocompatibility analysis in vivo

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