Use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in atherosclerosis

ABSTRACT

Dysfunction in the macrophage lysosomal system including reduced acidity and diminished degradative capacity is a hallmark of atherosclerosis, leading to blunted clearance of excess cellular debris and lipids in plaques and contributing to lesion progression. Devising strategies to rescue this macrophage lysosomal dysfunction is a novel therapeutic measure. Nanoparticles have emerged as an effective platform to both target specific tissues and serve as drug delivery vehicles. In most cases, administered nanoparticles are taken up non-selectively by the mononuclear phagocyte system including monocytes/macrophages leading to the undesirable degradation of cargo in lysosomes. We took advantage of this default route to target macrophage lysosomes to rectify their acidity in disease states such as atherosclerosis. Herein, we develop and test two commonly used acidic nanoparticles, poly-lactide-co-glycolic acid (PLGA) and polylactic acid (PLA), both in vitro and in vivo. Our results in cultured macrophages indicate that the PLGA-based nanoparticles are the most effective at trafficking to and enhancing acidification of lysosomes. PLGA nanoparticles also provide functional benefits including enhanced lysosomal degradation, promotion of macroautophagy/autophagy and protein aggregate removal, and reduced apoptosis and inflammasome activation. We demonstrate the utility of this system in vivo, showing nanoparticle accumulation in, and lysosomal acidification of, macrophages in atherosclerotic plaques. Long-term administration of PLGA nanoparticles results in significant reductions in surrogates of plaque complexity with reduced apoptosis, necrotic core formation, and cytotoxic protein aggregates and increased fibrous cap formation. Taken together, our data support the use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in the treatment of atherosclerosis.

Abbreviations: BCA: brachiocephalic arteries; FACS: fluorescence activated cell sorting; FITC: fluorescein-5-isothiocyanatel; IL1B: interleukin 1 beta; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; LSDs: lysosomal storage disorders; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MPS: mononuclear phagocyte system; PEGHDE: polyethylene glycol hexadecyl ether; PLA: polylactic acid; PLGA: poly-lactide-co-glycolic acid; SQSTM1/p62: sequestosome 1

Introduction

Disorders of the cellular lysosomal machinery have long been associated with a series of childhood genetic diseases known as lysosomal storage disorders (LSDs) [1–7]. Mutations in nearly 50 different lysosomal genes affect a diverse array of lysosomal functions related to the degradation and recycling of lipids, proteins, and sugars [8–12]. Clinical manifestations of these diseases include liver dysfunction, heart failure, and neurodegeneration. More recently, it is becoming increasingly appreciated that many common adult-onset diseases develop via progressive lysosomal dysfunction in ways that mimic the pediatric genetic disorders. These so-called “acquired” LSDs often lead to buildup of cargo targeted for lysosomal degradation and ensuing cellular/organ dysfunction [13–15].

One of the prototypical acquired lysosomal storage diseases is atherosclerosis. During lesion development, monocytes are recruited to the vascular subintima and differentiate into macrophages to clear retained lipids and cellular debris [16]. However, progressive uptake of poorly hydrolysable modified lipids, such as oxidized low-density lipoproteins (oxLDL), lead to accumulation of undigested or partly digested cholesteryl esters and free cholesterol within lysosomes [17]. This results in functional consequences including compromised processing of cargo and effects on associated processes such as blockage of autophagy and autophagy-lysosome fusion, leading to an inability to degrade deleterious organelles and protein aggregates [18,19]. A common concomitant occurrence in acquired LSDs such as atherosclerosis is the inability of lysosomes to maintain an acidic pH. Macrophages loaded with modified lipids lose lysosomal acidity, a process that appears to involve inhibition of vacuolar proton pumps (v-ATPases), thus, rendering lysosomes incapable of generating the required H⁺-gradient to maintain acidity [20,21]. Given the optimal function of most lysosomal enzymes occurs at acidic pH, perturbations in H⁺-gradients would disrupt critical enzymatic functions including lipid hydrolysis and other degradation processes including proteolysis (leading to undigested protein aggregates) [20,22–24].We and others have attempted to rescue the lysosomal dysfunction seen in atherosclerotic macrophages to assess if this could be leveraged as an atheroprotective measure. TFEB (transcription factor EB), a member of the MiT/TFE helix–loop–helix subfamily, is considered a transcriptional master regulator of lysosomal biogenesis with the ability to coordinate the upregulation of numerous lysosomal enzymes as well as several V-ATPases which regulate lysosomal acidification [25–27]. Indeed, macrophage-specific TFEB transgenic mice significantly increase lysosomal acidification and lysosomal enzyme expression resulting in enhanced macrophage degradative capacity and reduced atherosclerosis [28,29]. The degree to which correction of lysosomal acidity curtails the dysfunction in atherosclerotic macrophages remains unknown but is clearly an attractive therapeutic option. Additionally, it can be a strategy to recover the lysosomal pH of numerous other diseases which have lysosomal dysfunction as a prominent feature. Thus, we set out to create a context responsive nanoparticle system that could act to acidify lysosomal compartments and buffer the relatively low pH of these organelles.

Nanoparticulate agents (with sizes ranging from 1 to 300 nm) are readily available and several are currently approved for clinical use [30–32]. Polymeric nanoparticles are particularly attractive because of their well-defined size, surface charge, hydrophilicity, shape adaptability, and available functionality. In particular, polymeric nanoparticles such as PLGA and PLA, are commonly used due to numerous advantages including biocompatibility, biodegradability, ease of modification for interaction and targeting, and sustained/flexible release of drugs [33–35]. PLGA, which is considered one of the most promising polymeric nanoparticles for clinical use, is known to be taken up by cells through endocytic pathways, making it an attractive system to target lysosomes [36,37]. Given that hydrolysis of PLGA and PLA leads to production of glycolic and/or lactic acid, we became interested in the use of such nanoparticles to both target lysosomes and functionally rescue their lysosomal acidity.

In this manuscript, we provide evidence for the use of PLGA-based nanoparticles as an ideal lysosomal delivery vehicle which can restore lysosomal pH. In a series of experiments in primary macrophages in vitro, we first demonstrate that PLGA-based (as opposed to nonacidic control and PLA) nanoparticles are internalized by macrophages, enrich within lysosomes, and buffer/maintain lysosomal acidification. We then show that PLGA nanoparticles are effective at rescuing the autophagy-lysosome degradative capacity of macrophages and reducing their propensity to undergo apoptosis and inflammasome activation in the presence of lysosome-disrupting atherogenic lipids. Finally, we show that nanoparticles administered to mice, concentrate within both circulating myeloid cells and tissue macrophages in vivo. These tissue beds include the spleen, which sees heavy immune cell traffic, and atherosclerotic plaques, which are insidiously populated by macrophages. Most importantly, PLGA-based nanoparticles can acidify lysosomes within atherosclerotic plaque macrophages in vivo resulting in significant reductions in plaque complexity. Our study provides the first insights into developing a unique nanoparticulate system which acts to acidify lysosomes rendered dysfunctional by acquired LSDs such as atherosclerosis with tremendous therapeutic utility.

Results

Synthesis and characterization of control, PLGA- and PLA-based acidic nanoparticles

Nanoparticle systems have been developed to fulfil many desired characteristics encompassed in single-unit delivery agents including high surface area, enhanced loading capacity, reduced reactivity to biological systems, and targeting to desired sites [38,39]. Despite significant progress, premature release of cargo from nanoparticles remains a major drawback. Core-shell nanoparticle systems have been introduced as one of the solutions [40–44]. We desired to leverage the advantages of such a system to develop core-shell nanoparticles that could induce a low pH environment in biological systems for therapeutic application (Figure S1A). An important criterion to achieve was the need for these nanoparticles to gradually release their acid content. Thus, we prepared several nanoparticle samples as control or loaded with PLA, PLGA, and a combination of both PLA+PLGA with or without fluorescein-5-isothiocyanate (FITC)-labeling to enable tracking of these nanoparticles in cells and in vivo. The design of these core-shell nanoparticles included PLA and/or PLGA serving as the acidifying agent and a secondary polymer to stabilize the colloidal suspension. These nanoparticles were synthesized by a solvent evaporation method (see Table 1 in Material & Methods section). In order to control the size, the core-shell nanoparticles were co-self-assembled in the presence of a secondary amphiphilic agent, i.e., polyethylene glycol hexadecyl ether (PEGHDE) or an amphiphilic di-block copolymer (polystyrene-b-polyacryclic acid). The hydrodynamic diameter determination for these acidic nanoparticles showed a unimodal distribution ranging from ~60-150 nm, with PLA generally being smaller than PLGA followed by combined PLA+PLGA nanoparticles (Figure S1B).

Table 1.

Composition of nanoparticles developed using PEGHDE, PS-b-PAA, PLA, PLGA, and FITC.

	PEGHDE (mg/mL)	PLA (mg/mL)	PLGA (mg/mL)	PS-b-PAA (mg/mL)	Fluorescent tag (5 mol% of PS-b-PAA)
Control	0.5	-	-	0.5	-
PLA	0.5	1	-	0.5	-
PLGA	0.5	-	1	0.5	-
PLA + PLGA	0.5	1	1	0.5	-
Control (FITC-labeled)	0.5	-	-	0.5	FITC
PLA (FITC-labeled)	0.5	1	-	0.5	FITC
PLGA (FITC-labeled)	0.5	-	1	0.5	FITC
PLA + PLGA (FITC-labeled)	0.5	1	1	0.5	FITC

To study the ability of these nanoparticle samples to release the loaded PLA, PLGA, or combined PLA+PLGA, particle destabilization was assessed by measurement of change in hydrodynamic diameter in solutions of varying pH. Nanoparticles were incubated at near-neutral (pH 6.8) or acidic (pH 4.6) conditions at 37°C for 4 days and % changes in hydrodynamic diameter were evaluated every 2 days (Figure S1C, D). All acidic nanoparticle types were relatively stable in the first 48 h and even up to 4 days of incubation at near-neutral (pH 6.8) conditions (Figure S1C). As would be expected, we observed loss of integrity for all nanoparticles within the first 48 h under acidic conditions (pH 4.6) similar to what would be found in the lysosomal compartment of cells (Figure S1D).

Macrophages uptake nanoparticles in the lysosomal compartment with slow decay kinetics

To analyze the kinetics of nanoparticle uptake by macrophages in vitro, bone marrow derived macrophages (BMDMs) were incubated for various times with nanoparticles labeled with the fluorochrome FITC and imaged by live microscopy (Figures 1(a,b)). Over a short time-course, there was rapid uptake of nanoparticles beginning at 30 min and progressively accumulating up to 120 min (Figure 1(a), Figure S2). Using flow cytometry, we next analyzed nanoparticle uptake over a longer 12-h timeframe. As seen with live microscopy, FITC-labeled nanoparticles were internalized rapidly in the first 2–4 h, but uptake largely saturated from 4 to 12 h (Figure 1(b)). Fluorescence microscopy analysis at the 6-h time-point showed that the nanoparticles were predominantly endocytosed and persisted in the lysosomal (LAMP2⁺) system (Figure 1(c)).

Figure 1.

Macrophages uptake nanoparticles in the lysosomal compartment with slow decay kinetics. (a) Real-time live fluorescence microscopy of macrophages treated with FITC-labeled control nanoparticles for 120 min (images interrogated every 12 min). Quantification represents data from n = 20 cells. (b) FACS analysis of the uptake of FITC-labeled control nanoparticles by macrophages in culture during short (A) and long (B) time courses (minutes and hours indicated on the x-axis). Mean fluorescence intensity (MFI) of FITC was quantified from n = 3 independent experiments. (c) Immunofluorescence microscopy analysis acidic nanoparticle uptake by macrophages and their colocalization with lysosomes (LAMP2⁺). Representative images shown on left and quantification of the nanoparticle/LAMP2 colocalization at indicated times shown on right. (d) FACS analysis of the fluorescence decay after uptake of acidic nanoparticles by macrophages. MFI of FITC in macrophages was quantified from three independent experiments. (e) Quantification of the colocalization of nanoparticles with lysosomes (LAMP2⁺) with or without lysosomal inhibitor (chloroquine) to abort degradation (n ≥ 20 cells.) Representative images shown on left. For all graphs, data presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

To determine the longevity of nanoparticles while internalized, we incubated macrophages for 2 h with FITC-labeled nanoparticles as above followed by a wash-out period and serial quantification by fluorescence-activated cell sorting (FACS) analysis. The nanoparticles exhibited a slow decay over 24 h of wash-out with FITC fluorescence gradually decreasing to ~60% original intensity (Figure 1(d)). Nanoparticle degradation (gauged by progressive loss of fluorescence) occurred in the lysosomal system and was contributed to by lysosomal acidity as co-incubation of cells with chloroquine to disrupt lysosomal acidification significantly blunted nanoparticle degradation (Figure 1(e)).

PLGA-based nanoparticles are effective at maintaining lysosomal acidification in macrophages

Having determined the kinetics of our nanoparticles in macrophages, we desired to compare the efficiency of the three different types of nanoparticles in lysosomal acidification.

We incubated macrophages with control, PLA-, PLGA-, and PLA-PLGA-based nanoparticles and used the commonly used lysosomal pH-sensor LysoTracker Red as a readout. Over a 6-htime-course, only the PLGA- and PLA-PLGA-based nanoparticles enhanced LysoTracker intensity in macrophages, whereas PLA-based nanoparticles did not alter lysosomal acidification (Figure 2(a)). The equivalence of the PLGA- and PLA-PLGA-based nanoparticles suggested that it is the PLGA content (and not the PLA) of nanoparticles which is mediating acidification. Furthermore, evaluation of nanoparticle uptake tendency by macrophages showed no differences between control, PLA, and PLGA in regard to nanoparticle intake (Figure S3B), indicating that differences in nanoparticle acidification capacity does not affect intake efficiency. Additionally, escalation of the nanoparticle concentration in the media continued to demonstrate the superiority of PLGA-based nanoparticles over all other types including the PLA-PLGA-nanoparticles (Figure 2(b), Figure S3A). We next evaluated the length of time PLGA-based nanoparticles could maintain lysosomal acidity by incubating macrophages for 2 h with the nanoparticles followed by a wash-out period and serial quantification of LysoTracker Red by FACS analysis. Lysosomal acidity was largely maintained with approximately 70% of LysoTracker Red intensity remaining for up to 24 h (Figure 2(c)).

Figure 2.

PLGA-based nanoparticles are effective at maintaining lysosomal acidification in macrophages. (a,b) Analysis of macrophage lysosomal acidity by FACS after incubation with (A) low (20 μg/ml) or (B) medium (50 μg/ml) concentrations of control, PLA-, PLGA-, and PLA-PLGA-based nanoparticles for the indicated times. Mean fluorescence intensity (MFI) of LysoTracker Red was quantified from n = 3 independent experiments. (c) FACS analysis of the lysosomal acidity decay after uptake of PLGA nanoparticles by macrophages. MFI of LysoTracker Red quantified from n = 3 independent experiments. (d) FACS analysis of macrophage lysosomal acidity after incubation with lysosomal pH disruptor bafilomycin A₁ with or without either low (20 μg/ml) or medium (50 μg/ml) concentrations of PLGA nanoparticles. MFI of LysoTracker Red was quantified from n = 3 independent experiments. (e) Real-time live fluorescence microscopy of macrophages treated with LysoTracker Red and the lysosomal pH disruptor bafilomycin A₁ with either vehicle, control nanoparticles, or PLGA-based nanoparticles for 120 min (images interrogated every 12 min). Quantification represents data from n = 20 cells. (f) FACS analysis of macrophage lysosomal acidity after incubation with bafilomycin A₁ plus either control nanoparticles or PLGA-based nanoparticles for 0, 3,6,12 h. For all graphs, data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

In order to test the capacity of PLGA-based nanoparticles to buffer and maintain lysosomal acidity in the setting lysosomal pH disruptors, we incubated LysoTracker Red-labeled macrophages with the lysosomal proton-pump inhibitor bafilomycin A₁ and escalating doses of control versus PLGA nanoparticles. Bafilomycin A₁ abrogated lysosomal acidity and although low doses (20 ug/ml) of PLGA nanoparticles only marginally reversed this effect, higher concentrations (50 ug/ml) nearly completely rescued the loss of acidity (Figure 2(d)). We conducted parallel experiments using real-time live imaging to understand the kinetics of this rescue in lysosomal pH. Whereas control nanoparticles had no effect on bafilomycin A₁-induced abrogation of lysosomal acidity, PLGA-based nanoparticles were able to maintain lower lysosomal pH for the duration of the 2-h time-course (Figure 2(e), Figure S3E). In corroboration of the ability of PLGA-based nanoparticles to maintain lysosomal acidity for several hours (Figure 2(c)), we noted that amplification of lysosomal acidity was sustained for up to 12 h even in the presence of bafilomycin A₁ (Figure 2(f)). The protracted effects of PLGA-based nanoparticles were attractive for its use in vivo to alter monocyte/macrophage lysosomal pH.

PLGA-based nanoparticles can rescue macrophage autophagy-lysosomal dysfunction and ameliorate downstream sequelae

We next conducted a series of experiments to evaluate the effect of PLGA-based nanoparticles on macrophage phenotypes rendered dysfunctional by lysosomal disruptors, such as bafilomycin A₁, or more pathophysiologically-relevant atherogenic lipids, such as cholesterol crystals. First, we assessed the effect of PLGA nanoparticles on lysosomal proteolysis using fluorochrome-conjugated ovalbumin (DQ-ovalbumin), which is a fluorogenic protein substrate for proteases that is readily endocytosed to the lysosomal compartment and fluoresces upon degradation. Macrophages incubated with PLGA-based nanoparticles had enhanced ovalbumin proteolysis (Figure 3(a)). Additionally, protein degradation was clearly dependent on lysosomal acidity as bafilomycin A₁ disrupted ovalbumin proteolysis, which was completely rescued by the presence of PLGA nanoparticles (Figure 3(b)). A functional lysosome is a prerequisite to the process of autophagy wherein autophagic cargo is degraded by fusion with lysosomes upon pro-degradative stimuli such as nutrient-starvation [45]. Macrophages treated with bafilomycin A₁ followed by nutrient starvation demonstrated increased numbers of autophagosomes (as gauged by MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha) intensity and formation of LC3 puncta) and the autophagy chaperone SQSTM1/p62 (sequestosome 1) as a result of inhibited autophagy flux (Figure 3(c,d)). PLGA-based nanoparticles completely abrogated this inhibitory effect of bafilomycin A₁ on the progression of autophagy (Figure 3(c,d)). We buttressed this observation by also showing that PLGA nanoparticles can nearly completely rescue accumulation of SQSTM1-enriched/ubiquitin-positive protein aggregates in bafilomycin A₁-treated macrophages (Figure 3(e,f)). These results demonstrate that the autophagy-lysosome dysfunction instigated by bafilomycin A₁-dependent disruption of lysosomal acidity can be reversed by PLGA nanoparticles.

Figure 3.

PLGA-based nanoparticles enhance the autophagy-lysosomal degradative capacity of macrophages rendered dysfunctional by bafilomycin A_1. (a,b) Macrophages were pre-treated with 50 μg/ml of control or PLGA-based nanoparticles for 4 h followed by loading with (A) DQ-ovalbumin (10 μg/m) alone or (B) DQ-ovalbumin with and without bafilomycin A₁ (20 nM). FACS was used to quantify the mean fluorescence intensity (MFI) of DQ-ovalbumin for n = 3 independent experiments. (c,d) Macrophages were pre-treated with 50 μg/ml of control or PLGA-based nanoparticles for 4 h with or without bafilomycin A₁ (20 nM) followed by 30 min of nutrient starvation with HBSS to induce autophagy. Autophagy flux was quantified by (C) the autophagy marker LC3 (intensity and number of puncta) using immunofluorescence (IF) microscopy for n = 3 independent experiments (n ≥ 50 cells per group) and (D) levels of the autophagy chaperone SQSTM1 by western blot analysis of n = 3 independent experiments. (e,f) Macrophages were co-incubated with 50 µg/ml of control or PLGA-based nanoparticles with or without bafilomycin A₁ (20 nM) for 12 h and SQSTM1-enriched protein aggregates imaged using IF microscopy. Representative images of cells co-stained with SQSTM1 and polyubiquitin (FK1) antibodies shown on left (E) and quantification of SQSTM1 and ubiquitin intensity and SQSTM1⁺ ubiquitin⁺ aggregates (F) shown on right for n = 3 independent experiments (n ≥ 40 cells per group). For all graphs, data are presented as Mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

In order to determine whether PLGA nanoparticles can also rescue macrophage dysfunction promoted by lysosome-disrupting atherogenic lipids, we conducted similar experiments in macrophages incubated with cholesterol crystals, which are well-known to disrupt lysosomal acidity and function in atherosclerotic macrophages and resultant inflammasome-IL1B (interleukin 1 beta) activation and apoptosis [45–47]. Incubation of macrophages with cholesterol crystals resulted in significant accumulation of SQSTM1-enriched/ubiquitin-positive protein aggregates which were nearly completely resolved in the presence of PLGA nanoparticles (Figure 4(a,b)). Additionally, PLGA-based nanoparticles markedly reduced cholesterol crystal-induced apoptosis in macrophages (Figure 4(c)) and IL1B secretion in macrophages primed with lipopolysaccharide (LPS) whose inflammasome system was activated by cholesterol crystals (Figure 4(d)). Taken together, these results demonstrate that correction and buffering of lysosomal acidity by PLGA-based nanoparticles is beneficial and cytoprotective in macrophages whose lysosome system has been disrupted.

Figure 4.

PLGA-based nanoparticles ameliorate atherogenic lipid-induced lysosomal dysfunction and downstream sequela in macrophages. (a,b) Macrophages were co-incubated with 50 µg/ml of control or PLGA-based nanoparticles with or without cholesterol crystals (CC, 500 µg/ml) for 12 h and SQSTM1-enriched protein aggregates imaged using IF microscopy. Representative images of cells co-stained with SQSTM1 and polyubiquitin (FK1) antibodies shown on left (A) and quantification of SQSTM1 and ubiquitin intensity and SQSTM1/ubiquitin⁺ aggregates shown on right (B) for n = 3 independent experiments (n ≥ 40 cells per group). (c) Macrophages were co-incubated with treated with CC for 24 h and apoptosis evaluated by IF microscopy of CASP3-CASP7. Representative images of CASP3-CASP7 and nuclear stain (DAPI) shown on left and quantification of %positive cells shown on right (n = 3 independent experiments, n ≥ 400 cells per group). (d) Macrophages were co-incubated with 50 µg/ml of control or PLGA-based nanoparticles with LPS (100 ng/ml) and CC for 24 h to activate inflammasome signaling and ELISA of culture media used to measure secreted IL-1β (n = 3 independent wells). For all graphs, data are presented as mean ± SEM. **P < 0.01 and ***P < 0.001.

PLGA-based nanoparticles have preferential uptake in circulating monocytes and tissue macrophages in vivo

The mononuclear phagocyte system (MPS) with robust endocytic/lysosomal degradative ability is one of the predominant ways by which debris and circulating particulate material is cleared from the bloodstream and tissues [48,49]. Thus, one of the challenges of targeted nanoparticle therapeutic strategies has always been avoidance of and mitigating clearance by the MPS [50,51]. Interestingly, the predilection of nanoparticles to mononuclear cells was advantageous given our desire to target circulating monocytes and tissue macrophages for lysosomal acidification. We first evaluated the distribution and uptake of control nanoparticles in immune cells by administering FITC-conjugated nanoparticles intravenously followed by isolation of immune cells from blood and the spleen over a 4-h time-course (Figure 5(a)). FITC fluorescence was observed in blood monocytes after 1 h of injection and was significantly increased in splenic macrophages at 4 h (Figures 5(b,c)). There was also increased uptake seen at 1 h in the other major myeloid lineage, granulocytes (Figures S4A, B). However, both circulating and splenic B-cells and T-cells were not targeted by the nanoparticles, suggesting that they have a specific affinity for the MPS and circulating/tissue myeloid cells (Figures 5(b,c) and Figures S4A,B).

Figure 5.

PLGA-based nanoparticles are effective at acidifying tissue macrophages in vivo. (a) Summary of experimental protocol for the in vivo evaluation of FITC-labeled nanoparticle uptake over a 4-h period in circulating and splenic immune cells (monocytes, neutrophils, B-cells, and T-cells). (b,c) FACS analysis of the uptake of FITC-labeled nanoparticles by blood monocytes (B) and splenic macrophages (C) for the indicated times. Mean fluorescence intensity (MFI) of LysoTracker Red was quantified for n = 3 independent experiments. (d) Flow cytometer analysis of the persistence of acidic nanoparticles in splenic macrophages at different time points after i.v. injection. MFI of LysoTracker Red in immune cells was quantified from n = 3 independent experiments. (e) Summary of experimental protocol for detecting splenic macrophage lysosomal acidification at 6 h after i.v. administration of control, FITC-labeled control, and FITC-labeled PLGA-based nanoparticles. (f-h) FACS analysis of FITC⁺ nanoparticles taken up by splenic macrophages (F), lysosomal acidification via LysoTracker Red co-staining (G), and lysosomal acidification via LysoTracker Red in FITC⁺ macrophages (H). MFI of LysoTracker Red was quantified from n = 3 independent experiments. For all graphs, data are presented as mean ± SEM. *P < 0.05, ***P < 0.001.

PLGA-based nanoparticles are effective at acidifying tissue macrophages in vivo

Having demonstrated that our nanoparticles are preferentially taken up by splenic macrophages, we next evaluated the acidifying potential of PLGA-based nanoparticles. We first conducted a longer-term experiment in vivo to determine the peak uptake of nanoparticles in splenic macrophages. Over a 12-h time-course, FITC-labeled nanoparticles injected intravenously in mice were observed in splenic macrophages as early as 3 h, peaking at 6 h, and were largely maintained up to 12 h (Figure 5(d)). We thus used 6-h as the time-point to evaluate the acidifying potential of the PLGA nanoparticles. A cohort of mice were intravenously administered control, FITC-labeled control, or FITC-labeled PLGA-based nanoparticles and lysosomal acidification assessed by LysoTracker Red staining and FACS analysis (Figure 5(e)). Although similar amounts of splenic macrophage were observed to be FITC⁺, only the group administered PLGA nanoparticles developed significantly greater LysoTracker staining (Figures 5(f-h)).

PLGA-based nanoparticles are taken up by and acidify the lysosomes of atherosclerotic plaque macrophages

Given that the spleen is a major organ for the circulation and trafficking of immune cells including myeloid cells, nanoparticle uptake in splenic macrophages is readily expected. We next desired to evaluate the extent to which nanoparticles are taken up by macrophages in atherosclerotic plaques, a tissue bed where nanoparticle therapeutics would be of utmost benefit. A cohort of atherogenic apoe^−/− mice were fed a Western diet for 8 weeks, a duration necessary for the development of significant plaque burden at the aortic root [52]. Each mouse was then administered FITC-conjugated nanoparticles or vehicle intravenously and nanoparticle uptake assessed at 24 h (Figure 6(a)). Microscopy imaging of aortic root sections demonstrated areas of green fluorescence suggestive of significant nanoparticle uptake over the 24-h period (Figure 6(b)). Further co-staining with the macrophage-specific antibody against CD68 demonstrated significant colocalization consistent with nanoparticle uptake in plaque macrophages (Figure 6(c)).

Figure 6.

PLGA-based nanoparticles are taken up by and acidify the lysosomes of atherosclerotic plaque macrophages. (a) Summary of experimental protocol for detecting i.v. injected FITC-labeled nanoparticles in macrophages from atherosclerotic plaques of apoe^−/− mice fed 8 weeks of Western diet. (b,c) Fluorescence microscopy of atherosclerotic aortic root sections from mice in (A) administered vehicle (n = 3) or FITC-labeled control (n = 3) nanoparticles and imaged after 24 h for the FITC label (B) or co-stained with macrophage marker CD68 (C). Representative figures are shown on left of each graph. (d) Summary of experimental protocol for detecting macrophage lysosome acidification in atherosclerotic plaques of apoe^−/− mice fed 8 weeks of Western diet and i.v. injected with FITC-labeled nanoparticles. (E) FACS analysis of macrophages isolated from atherosclerotic plaques from mice in (D) administered FITC-labeled control (n = 3) or FITC-labeled PLGA (n = 3) nanoparticles and stained with LysoTracker Red to determine lysosomal acidification. Mean fluorescence intensity (MFI) of LysoTracker Red in macrophages was quantified. For all graphs, data are presented as mean ± SEM. *P < 0.05, and ***P < 0.001.

In experiments similar to the splenic macrophages above, we intravenously administered FITC-labeled control or PLGA-based nanoparticles in cohorts of atherogenic apoe^−/− mice fed Western diet for 8 weeks and after 24 h analyzed LysoTracker Red-stained plaque macrophages by FACS (Figure 6(d)). The PLGA nanoparticles significantly elevated lysosomal acidity compared to the control counterpart (Figure 6(e)), suggesting that our PLGA-based system was effective at acidifying macrophage lysosomes in not only splenic macrophages but plaque macrophages.

PLGA nanoparticles improve atherosclerotic lesion by reducing apoptosis, alleviating cargo aggregation, and elevating plaque stability

To characterize the impact of PLGA nanoparticles on atherosclerotic lesion, apoe^−/− mice were fed a Western diet for 4 weeks, while control or PLGA nanoparticles were administered by intravenous injection at regular intervals (3 times per week) throughout this period (Figure 7(a)). Administration of PLGA nanoparticles during Western diet feeding led to intake and accumulation of nanoparticles in macrophages from both aortic roots and spleen (Figure S5A). As expected, uptake of PLGA nanoparticles significantly elevated lysosomal acidity in aortic macrophages compared to control (Figure 7(b)). While serum cholesterol concentration increased as a result of Western diet feeding, there were no differences between control and PLGA nanoparticle-treated mice (Figure 7(c)). Interestingly, a tendency toward decreased aortic root plaque burden (smaller, albeit not significant, lesion size) in mice receiving PLGA nanoparticles compared to control after 4 weeks of Western diet feeding (Figure 7(d)). Similarly, macrophage content within aortic root lesions, as gauged by MOMA-2 and CD68 staining, did not differ significantly (Figure 7(e) and Figure S5B), however, both plaque complexity (smaller area of acellular, necrotic debris) and apoptosis (less cells positive for cleaved-Caspase-3) were lessened in aortic roots from PLGA nanoparticle-treated mice compared to controls (Figure 7(f, g)). Similar results in plaque complexity parameters were seen within the brachiocephalic arteries (BCA) (Figure 7(h-j) and Figure S5B), indicating administration of PLGA nanoparticles efficiently improved atherosclerotic lesion stability, particularly in terms of inhibiting macrophage cell death and necrosis.

Figure 7.

Beneficial effects of acidic nanoparticles on rescuing macrophage dysfunction in atherosclerosis. (a) Summary of experimental protocol for detecting the improvement in atherosclerotic lesion by administration of PLGA nanoparticles in apoe^−/− mice fed 4 weeks of Western diet. (b) FACS analysis of macrophages isolated from atherosclerotic plaques from mice fed Western diet in administered control (n = 6) or PLGA (n = 6) nanoparticles and stained with LysoTracker Red to determine lysosomal acidification. Mean fluorescence intensity (MFI) of LysoTracker Red in macrophages was quantified. (c) Serum cholesterol of apoe^−/− mice before and after placement on Western diet for 4 weeks in administered control (n = 7) or PLGA (n = 6) nanoparticles. (d) Quantification of atherosclerotic plaque burden in Oil Red O–stained aortic root sections from apoe^−/− mice fed the Western diet for 4 weeks (Control: n = 7; PLGA: n = 6); representative roots are shown on the left, lesion areas are shown on the right. (e-g) Plaque composition quantified by immunofluorescence (IF) microscopy of aortic root sections for (E) macrophages (MOMA-2), (F) necrotic core, and (G) apoptosis (cleaved-Caspase-3⁺). Representative roots are shown on the left. (h-j) Plaque composition quantified by IF microscopy of brachiocephalic artery (BCA) sections for (H) macrophages (MOMA-2), (I) necrotic core, and (J) apoptosis (cleaved-CASP3⁺). Representative roots are shown on the left. For all graphs, data are presented as mean ± SEM. *P < 0.05, and **P < 0.01.

Our previous studies have shown increased cell death within atherosclerotic plaques closely correlates with macrophage lipid cargo accumulation and foam cell formation, which can be characterized by increased markers of degradation, namely SQSTM1 (autophagy cargo receptor) and polyubiquitinated protein aggregates [53]. Therefore, given the protective effect of PLGA nanoparticles against cell death in atherosclerotic plaques, we next assessed the effects on protein aggregation. Interestingly, compared to control, PLGA nanoparticles alleviated the accumulation of both SQSTM1 and polyubiquitinated protein within both aortic root (Figure 8(a)) and BCA lesions (Figure 8(b)). This finding was analogous to our results in cultured macrophages (Figure 4), and suggested PLGA nanoparticles prevent protein aggregation through augmented lysosomal function and aggrephagy, which in turn limits cell death and improves atherosclerotic lesion complexity. In addition, a significant increase in fibrous cap thickness was observed with PLGA administration (Figure 8(c)), which is an important feature denoting improved plaque stability. Previous studies have reported thinning of the fibrous cap to be highly correlated with foam cell infiltration, leading to plaque erosion and rupture [54]. Therefore, our study suggests PLGA nanoparticles efficiently improve plaque lesion environment via multiple mechanisms, leading to protection against atherosclerosis.

Figure 8.

PLGA nanoparticles alleviate cargo aggregation in atherosclerotic plaques and improve plaque stability. (a,b) Quantification of protein aggregation inside lesion area by immunofluorescence staining with antibodies against the ubiquitinated proteins (FK-1 antibody) and SQSTM1 in aortic roots (A) and brachiocephalic arteries (BCA) (B) from mice fed Western diet (Control: n = 7; PLGA: n = 6). Representative roots are shown on the left. (c) Quantification of fibrous cap area inside lesion area from aortic roots by immunofluorescence staining with antibodies against the SMC-1. Representative roots are shown on the left. For all graphs, data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Discussion

In this manuscript, we describe the development of an acidic nanoparticle delivery system which can concentrate in macrophage lysosomes and act to maintain the acidic pH necessary for lysosomal function. Amongst two commonly used acidic nanoparticles (PLA and PLGA), we find that PLGA-based nanoparticles are significantly better at lysosomal acidification in cultured macrophages and provide functional benefits to macrophages in the presence of atherogenic lipids. In the follow up studies in vivo, we demonstrate our nanoparticles target circulating myeloid cells and tissue macrophages including macrophages residing in the atherosclerotic plaques. Finally, we show that once taken up, PLGA-based nanoparticles are effective at buffering macrophage lysosomal acidity and ameliorating atherosclerotic plaque complexity including reduced cytotoxic protein aggregates, apoptosis, and necrotic core formation (Figure 9 summarizes these findings in pictorial format). Our work has significant implications in future disease therapeutics as PLGA nanoparticles can correct the loss of lysosomal acidity, which is seen in many forms of acquired lysosomal dysfunction including atherosclerosis. In addition to effects on pH, these nanoparticles can potentially be formulated to deliver drugs, proteins, or enzyme replacement therapies to dysfunctional lysosomes.

Figure 9.

Summary of the plaque stabilizing effects of acidic nanoparticles in atherosclerosis. Administration of acidic nanoparticles (aNP) can non-selectively target atherosclerotic plaque macrophages which are often lipid-laden with lysosomal dysfunction (i.e. the so-called macrophage foam cell). This can rescue lysosomal acidity and enable improved lysosomal function/autophagy flux resulting in clearance of cytotoxic protein aggregates, reduced macrophage apoptosis, and necrotic core formation (all surrogates of reduced plaque complexity/improved plaque stability).

Polymer nanoparticles have a longstanding history in the field of drug delivery and clinical therapeutics. Several previous papers have described methods of ameliorating atherosclerosis via nanoparticle-based drug delivery systems including the use of rapamycin-loaded PLGA nanoparticles cloaked with red blood cell membranes [55–57]. However, whether such nanoparticles are able to target atherosclerotic macrophages and alter lysosomal acidification is unknown. Our study is the first to develop and characterize PLA- and PLGA-based polymeric nanoparticles and to label them with fluorochrome, thus providing advantages for imaging and tracking the nanoparticles both in vitro and in vivo.

We observed rapid cellular uptake and colocalization of the nanoparticles with lysosomes, a significant amount of which were retained up to 24 h. We also identified PLGA nanoparticles as an efficient modulator of macrophage lysosomal acidity with an ability to rescue bafilomycin A₁-induced lysosomal dysfunction. These effects were also observed in vivo. After intravenous administration, acidic nanoparticles were taken up by splenic macrophages at 4 h and macrophages in atherosclerotic plaques after 24 h. Importantly, PLGA nanoparticles promoted further acidification of macrophage lysosomes in both the spleen and atherosclerotic plaques of mice, supporting the therapeutic utility of this nanoparticle system. In this regard, several issues are worthy of discussion.

Both PLA and PLGA have been shown to restore lysosomal acidity. In the APRE-19 (retinal pigment epithelia) cell-line, both PLA and PLGA nanoparticles were found to traffic to lysosomes, maintain lysosomal acidity, and elevate CTSD (cathepsin D) activity, although PLA nanoparticles did this more efficiently [58]. In primary macrophages, we find the opposite effect with PLGA nanoparticles having significantly superior effects on macrophage lysosomes, with PLA nanoparticles largely being ineffective. Although this difference could partly be due to differences in uptake efficiency and lysosomal trafficking of the nanoparticles, one possible reason may be the differences in elution efficiency between the PLA and PLGA nanoparticles in acidic conditions, a scenario particularly relevant to cells such as the macrophage with robust degradative capacity requiring an abundant lysosomal system.

One of the critical challenges in nanoparticle delivery systems is avoidance of 1) uptake by the mononuclear phagocyte system and 2) trafficking and degradation by the endolysosomal system [59–61]. MPS-mediated lysosomal degradation of therapeutic nanoparticles reduces both the appropriate targeting to desired cells as well as the dose needed for therapeutic efficacy. The significant advantage of our proposed PLGA nanoparticle system is that we have shown delivery to and concentration within tissue macrophages, which occurs primarily via passive targeting and default trafficking to lysosomes (i.e., therapeutic target). Thus, none of the challenges traditionally faced by nanoparticle design are operational in the delivery of acidic nanoparticles to tissue macrophage lysosomes. However, it is likely that one can improve the degree of targeting to tissue macrophages, particularly to specific subsets within atherosclerotic plaques with lysosomal dysfunction. There are various reports of nanoparticle modification to improve macrophage targeting.

PLGA nanoparticles do not solely target the MPS and macrophages. Oral administration of PLGA-based nanoparticles have also been shown to accumulate in the liver, kidney, and even brain [62]. Decoration of nanoparticles with affinity ligands/probes can improve binding to surface receptors of select cells. These include use of viral macrophage inflammatory protein-II (vMIP-II), SCARB1/CD36 ligand, LyP-1 peptide, mannose binding receptor ligands, ferritin, HDL-based particles to improve macrophage targeting [63–68]. More selective targeting to macrophages of the atherosclerotic plaque can leverage cell surface markers overexpressed in plaque macrophages such as hyaluronic acid-conjugated nanoparticles to foster binding of CD44-expressing macrophages abundantly found in atherosclerotic plaques [69]. Whether such strategies significantly improve PLGA nanoparticle delivery to macrophages compared to passive uptake by the macrophage phagolysosomal system will have to be determined.

In addition to defects in the lysosomal acidity of plaque macrophages, there is often dysfunction in critical lysosomal enzymes involved in cargo degradation. LIPA/LAL (lipase A, lysosomal acid type) for example is an especially critical enzyme required for efficient hydrolysis of cholesteryl esters and triglycerides being delivered extracellularly via lipoproteins or intracellularly via lipophagy [70]. Much evidence suggests dysfunction in the LIPA activity of plaque macrophages resulting in the development of undigested lipids and foam cell formation akin to genetic LIPA-deficiencies such as Wolman disease [71]. This so-called acquired lysosomal storage disease is an important contributor to the pathogenesis of atherosclerosis. Nanoparticle delivery of excess LIPA or other lysosomal enzymes to lysosomes for enhanced degradation of lipids and other lysosomal contents would be advantageous. Future design iterations of our PLGA nanoparticles to “cage” lysosomal enzymes could provide an ideal therapeutic platform by providing combined lysosomal acidification and enzyme hydrolysis to cells with a dysfunctional lysosomal system. Further fine-tuning to allow for slow elution of the PLGA to first acidify lysosomes followed by enzyme release could provide a highly effective lysosomal enzyme replacement therapy for chronic diseases such as atherosclerosis.

Materials and methods

Animals

Animal protocols were approved by the Washington University Animal Studies Committee. All mice used in this study were either C57BL/6 J strain or apoe^−/− mice on C57BL/6 J background (>N7). Mice housed in a specific pathogen–free barrier facility were weaned at 3 weeks of age to a standard mouse chow providing 6% calories as fat. For in vivo experiments, apoe^−/− male mice were started at ~8 weeks of age with standard Western-type diet (Harlan, TD 88137): 0.15% cholesterol, 42% calories as fat, 15% calories as protein [52]. For in vitro experiments, macrophages derived from mice aged between 2 to 6 months were used and within each experiment only mice with the same sex and similar age were compared.

Synthesis of acidic nanoparticles

A core shell nanoparticle synthesis method was used in preparing all the acidic nanoparticles along with required controls [40]. Nanoparticles were prepared by solvent-evaporation method while using polyethylene glycol hexadecyl ether (PEGHDE; Sigma Aldrich) as core of the particle. In a typical procedure, a 0.5 mg/mL of PEGHDE aqueous solution was prepared by melting 2.5 mg of PEGHDE at 60°C for 10 min in a 25 ml glass sample viol. A 5-mL volume of autoclaved MilliQ water was added to the viol while stirring the mixture at 110 x g. After 10 min of incubation, PLGA, PLA, or both dissolved in THF (1 mg in 250 µL of THF) were added to aqueous solution of PEGHDE. Immediately after this addition a THF solution of PS-b-PAA (PS-b-PAA (polydispersity index: PDI = 1.18, 0.0033 mmoles) 0.5 mg in 250 µL of THF) was added to the stirring mixture drop-by-drop (one drop per ten seconds using a 22-gauge syringe). All the samples were prepared with or without fluorescence tag with FITC as described in Table 1.

Macrophage culture and treatment

Standard techniques were used to isolate thioglycollate-elicited peritoneal macrophages and bone marrow-derived macrophages. Briefly, mice were injected with 4% sterile thioglycollate media (Sigma Aldrich, T9032) intraperitoneally, and 4 days later, peritoneal macrophages were collected, counted and plated (DMEM [Sigma Aldrich, D5796] with 10% fetal bovine serum). Where indicated, the following treatments were performed on macrophages: chloroquine (Sigma Aldrich, C6628; 10 nM), and bafilomycin A₁ (Sigma Aldrich, B1793; 100 nM). Treated cells were harvested at various times for stained with antibodies for FACS analysis or fixed with 4% paraformaldehyde for immunofluorescence microscopy.

Flow cytometric analyses

Fluorescence activated cell sorting (FACS) analysis for nanoparticles uptake was performed on cultured macrophages and splenic macrophages. Cultured macrophages were plated (Greiner Bio-One, 665,102), treated with the indicated nanoparticles. Cells were collected and resuspended in FACS buffer (2% FBS in PBS [Life Technologies, 14,190,235]) for subsequent flow cytometry. For splenic macrophages, after injection with nanoparticles upon time indicated, dissected spleens were minced and prepared for single cell suspensions using a 70-μm cell strainer. Red cells were removed with Red Cell Lysis Buffer (155 mM NH₄Cl, 12 mM NaHCO₃, 0.1 mM EDTA). Cells were then labeled with Pacific Blue-conjugated CD45 (BioLegend, 103,126; 1:200), FITC-conjugated ADGRE1/F4/80 (BioLegend, 123,108; 1:200), and PerCP-Cy5.5-conjugated ITGAM/CD11b (BioLegend, 101,228; 1:200) antibodies on ice for 30 min, washed, and resuspended in FACS buffer for analysis.

FACS analysis for lysosomal activity was performed on cultured macrophages, splenic macrophages, and macrophages derived from atherosclerotic mouse aortas. Cultured macrophages were plated (Greiner Bio-One, 665,102), treated with the indicated reagents, and incubated with LysoTracker Red (Life Technologies, M7514; 200 nM) at 37°C for 30 min. Cells were collected and resuspended in FACS buffer (2% FBS in PBS) for subsequent flow cytometry. For splenic macrophages, dissected spleens were minced and prepared for single cell suspensions using a 70-μm cell strainer. Cells were washed with FACS buffer then incubated with LysoTracker Red at 37°C for 30 min. Cells were then labeled with antibodies stated above on ice for 30 min, washed, and resuspended in FACS buffer for analysis. For aortic macrophages, dissected/cleaned aortas (extending from the aortic root to the abdominal aorta at the level of the renal arteries) were incubated at 37°C for 60 min in a digestion buffer consisting of RPMI, 2.5 µg/mL Liberase (Roche, 05401127001), 125 µg/mL DNAse 1 (Sigma Aldrich, D4527), and 0.8 mg/mL hyaluronidase (Sigma Aldrich, H3506). Single cell suspensions were prepared using a 70-µm cell strainer and incubated with LysoTracker Red at 37°C for 30 min. Cells were then labeled with antibodies stated above on ice for 30 min, washed, and resuspended in FACS buffer for analysis.

FACS analysis for the assessment of lysosomal proteolysis was performed on cultured macrophages. Cells were plated and treated with nanoparticles (50 µg/mL) plus bafilomycin A₁ (20 nM) for 4 h. Two hours before collection, media was replaced with fresh media containing 10 µg/mL DQ-ovalbumin (Life Technologies, MP12053). Cells were then collected and analyzed by FACS as above.

FACS analysis for bio-distribution of nanoparticles in vivo was performed as follows: 2-month-old C57BL/6 J mice were given 200 μl nanoparticles (8 mg/kg) by intravenous injection and then dissected after time indicated. Blood and spleen were collected for single cell suspensions prepared as shown above. Cells were then labeled with Pacific Blue-conjugated CD45 (BioLegend, 103,126; 1:200), FITC-conjugated ADGRE1/F4/80 (BioLegend, 123,108; 1:200), PerCP-Cy5.5-conjugated ITGAM/CD11b (BioLegend, 101,228; 1:200), Alexa Fluor 700-conjugated LY6C (BioLegend, 128,024; 1:200), PE/Cy5-conjugated GSR/Gr-1 (BioLegend, 108,410; 1:200), BV605-conjugated CD19 (BioLegend, 115,539; 1:200), and PE/Cy7-conjugated CD3 (BioLegend, 100,219; 1:200) antibodies on ice for 30 min, washed, and resuspended in FACS buffer for analysis. All samples were analyzed using the BD Biosciences Canto II or LSR II flow cytometer and quantified using FlowJo software.

Immunofluorescence microscopy

Immunofluorescence (IF) microscopy of macrophages and frozen-tissue sections was performed as previously described [53]. Briefly, cells or tissues were fixed with 4% paraformaldehyde, blocked and permeabilized (1% BSA, 0.2% milk powder, 0.3% Triton X-100 [Sigma Aldrich, X100] in TBS (20 mM Tris, 10 mM NaCl, pH 7.4), and incubated with the antibodies sequentially. Specificity of staining was tested in control experiments either by omitting primary antibodies or using samples from knockout mice where available. The following primary antibodies were used at 1:250 dilution: LC3 (MBL International Corporation, PM036), SQSTM1/p62 (Progen Biotechnik, GP62-C), poly-ubiquitinated proteins (FK-1; Enzo Life Sciences, BML-PW8805), LAMP2 (Abcam, ab13524) and CD68 (Bio-Rad Laboratories, MCA1957). Species-specific fluorescent secondary antibodies were obtained from Invitrogen/Life Technologies (used at 1:250 dilution). CellEvent Caspase-3/7 Green Detection Reagent (Life Technologies, C10423) was used according to the manufacturer’s protocol. A Zeiss LSM-700 confocal microscope was used for image acquisition and images quantified using ZEN microscope software (Carl Zeiss AG).

Western blotting

Cells were lysed in a standard RIPA lysis buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.2, 0.1% Triton X-100, 1% sodium deoxycholate [Sigma Aldrich, D6750], 5 mM EDTA) containing protease inhibitor cocktail (Sigma-Aldrich, 4,693,132,001) and phosphatase inhibitors (Thermo Scientific, A32955) on ice. Lysed samples were centrifuged at 10,000 g for 10 min. Standard techniques was used for protein quantification, separation, transfer, and blotting. The following primary antibodies were used: SQSTM1/p62 (Abcam, ab56416; 1:1000), and ACTB/β-actin (Sigma Aldrich, A2066; 1:2000).

ELISA

For the assessment of atherogenic lipid-induced inflammatory cytokine IL1B, macrophages were plated and stimulated by LPS (100 ng/ml; Sigma Aldrich, A23831G) and cholesterol crystals (CC, 500 µg/ml; Sigma Aldrich, C3045) with and without 50 µg/ml nanoparticles. After 24 h, cell culture medium was collected and ELISA was performed as per the manufacturer’s protocols to detect secreted IL1B (R&D Systems, MLB00B).

Assessment of lysosomal acidity in macrophages by live imaging microscopy

For live imaging analysis of lysosomal acidity, macrophages were plated on glass-bottom culture dishes (Mattek Corporation, P35G-1.5–10-C). Macrophages were then treated with indicated reagents and live fluorescence imaging conducted using a Nikon A1Rsi Confocal Microscope with Tokai-hit stage-top incubator at 37°C and 5% CO₂. Drugs and nanoparticles were added after the first image, and images was captured every 12 min. Image analysis and quantification was performed using ImageJ software, regions of interest with thresholds were determined, and signal over threshold was quantified.

Quantification of nanoparticles at the aortic roots

Preparation of aortic root section from mice with atherosclerosis was as previously described [52]. Briefly, 2-month-old apoe^−/− mice were placed on a Western diet for 8 weeks to develop plaques followed by intravenous (i.v.) injection of nanoparticles (8 mg/kg) and dissected. PBS-perfused hearts were placed in a cryostat mold containing tissue freezing medium. Sections (10-µm thick) were taken from the samples beginning just caudal to the aortic sinus and extending into the proximal aorta. Slides were fixed with 4% paraformaldehyde and stained with anti-CD68 antibody (Bio-Rad Laboratories, MCA1957) followed with Alexa Fluor 594 goat anti-rat secondary antibody (Life Technologies, A11007). Images were taken by Zeiss LSM-700 confocal microscope and were quantified using ZEN microscope software (Carl Zeiss AG).

Evaluation of the effect of acidic nanoparticles on atherosclerotic lesion size and complexity

Two-month-old apoe^−/− mice were placed on a Western diet while concomitantly i.v. injected with FITC-labeled control or PLGA nanoparticles (8 mg/kg, 3x/week) starting one week after initiation of Western diet feeding. After 4 weeks, mice were dissected to collect aortic roots and brachiocephalic arteries (BCA) for frozen sections as described above. The ascending and thoracic portions of the atherosclerotic aortas were also harvested for the evaluation of lysosomal acidity in aortic macrophages by FACS. Quantification of atherosclerosis at the aortic root was carried out via Oil Red O (Sigma Aldrich, O0625) staining. Images were taken by EVOS XL Core Cell Imaging system and Oil Red O-positive regions were quantified using ZEN microscope software (Carl Zeiss AG). Characterization of plaque complexity was conducted by analysis for apoptosis, necrotic core size, SQSTM1-ubiquitin intensity, and fibrous cap of aortic root or BCA sections via immunofluorescence staining and confocal microscopy (Zeiss LSM-700) as previously described [72].

Statistical analyses

Statistical significance of differences was calculated using the Student’s unpaired t test or ANOVA (for multiple groups) followed by either Dunnett’s test (when multiple groups are compared with a single control) or Tukey’s multiple comparison test. Graphs containing error bars show the mean ± standard error of the mean (SEM). Statistical significance is represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, NS = not significant.

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Funding Statement

This work was supported by the National Institutes of Health [R01 HL125838]; National Institutes of Health [R01 DK121560]; National Institutes of Health [P30 DK056341]; National Institutes of Health [T32 HL134635]; u.s. department of veterans affairs [I01 BX003415], American Heart Association [897628].

Disclosure statement

D.P. is the founder/co-founder of three university start-ups. None of these entities, however, supported this work.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2022.2108252