In this account, we varied PEGylation density on the surface of hydrogel PRINT nanoparticles and systematically observed the effects on protein adsorption, macrophage uptake, and circulation time. Interestingly, the density of PEGylation necessary to promote a long-circulating particle was dramatically less than what has been previously reported. Overall, our methodology provides a rapid screening technique to predict particle behavior in vivo and our results deliver further insight to what PEG density is necessary to facilitate long-circulation.

Extended circulation of nanoparticles in blood is essential for most clinical applications. Nanoparticles are rapidly cleared by cells of the mononuclear phagocyte system (MPS). Approaches such as grafting polyethylene glycol onto particles (PEGylation) extend circulation times; however, these particles are still cleared, and the processes involved in this clearance remain poorly understood. Here, we present an intravital microscopybased assay for the quantification of nanoparticle clearance, allowing us to determine the effect of mouse strain and immune system function on particle clearance. We demonstrate that mouse strains that are prone to Th1 immune responses clear nanoparticles at a slower rate than Th2-prone mice. Using depletion strategies, we show that both granulocytes and macrophages participate in the enhanced clearance observed in Th2-prone mice. Macrophages isolated from Th1 strains took up fewer particles in vitro than macrophages from Th2 strains. Treating macrophages from Th1 strains with cytokines to differentiate them into M2 macrophages increased the amount of particle uptake. Conversely, treating macrophages from Th2 strains with cytokines to differentiate them into M1 macrophages decreased their particle uptake. Moreover, these results were confirmed in human monocyte-derived macrophages, suggesting that global immune regulation has a significant impact on nanoparticle clearance in humans. IntroductionThe potential clinical applications of nanoparticles and nanoformulations have been investigated for more than 30 years. Nanoparticle approaches have the potential to revolutionize drug delivery by allowing for the encapsulation of drugs with poor solubility or stability in a stable carrier particle. In addition, targeting nanoparticles to specific pathological sites may allow an increased effective dose of drug at the needed site, while decreasing systemic drug exposure, and therefore side effects. However, to date, only 2 nanoformulations for cancer treatment have been approved for clinical use (liposomal doxorubicin [Doxil] and protein-bound paclitaxel [Abraxane]) (1-3). One major obstacle for the use of nanoparticles in vivo is rapid clearance by the cells of the reticuloendothelial system (RES)/mononuclear phagocyte system (MPS) (4-7). In addition to rapid clearance, variable activity of the MPS among patients leads to widely variable pharmacokinetics of nanoformulations in the clinic, reducing the efficacy of both approved and future experimental nanoformulations (8).

Engineered nanoparticles have the potential to expand the breadth of pulmonary therapeutics, especially as respiratory vaccines. Notably, cationic nanoparticles have been demonstrated to produce superior local immune responses following pulmonary delivery; however, the cellular mechanisms of this increased response remains unknown. To this end, we systematically investigated the cellular response of lung APCS following pulmonary instillation of anionic and cationic charged nanoparticles. While nanoparticles of both surface charges were capable of trafficking to the draining lymph node and were readily internalized by alveolar macrophages, both CD11b and CD103 lung dendritic cell (DC) subtypes preferentially associated with cationic nanoparticles. Instillation of cationic nanoparticles resulted in the upregulation of Ccl2 and Cxc10, which likely contributes to the recruitment of CD11b DCs to the lung. In total, these cellular mechanisms explain the increased efficacy of cationic formulations as a pulmonary vaccine carrier and provide critical benchmarks in the design of pulmonary vaccine nanoparticles.

Pulmonary immunization enhances local humoral and cell-mediated mucosal protection, which are critical for vaccination against lung-specific pathogens such as influenza or tuberculosis. A variety of nanoparticle (NP) formulations have been tested preclinically for pulmonary vaccine development, yet the role of NP surface charge on downstream immune responses remains poorly understood. We used the Particle Replication in Non-Wetting Templates (PRINT) process to synthesize hydrogel NPs that varied only in surface charge and otherwise maintained constant size, shape, and antigen loading. Pulmonary immunization with ovalbumin (OVA)-conjugated cationic NPs led to enhanced systemic and lung antibody titers compared with anionic NPs. Increased antibody production correlated with robust germinal center B-cell expansion and increased activated CD4(+) T-cell populations in lung draining lymph nodes. Ex vivo treatment of dendritic cells (DCs) with OVA-conjugated cationic NPs induced robust antigen-specific T-cell proliferation with ∼ 100-fold more potency than soluble OVA alone. Enhanced T-cell expansion correlated with increased expression of surface MHCII, T-cell coactivating receptors, and key cytokines/chemokine expression by DCs treated with cationic NPs, which were not observed with anionic NPs or soluble OVA. Together, these studies highlight the importance of NP surface charge when designing pulmonary vaccines, and our findings support the notion that cationic NP platforms engender potent humoral and mucosal immune responses.

Long-circulating nanoparticles are essential for increasing tumor accumulation to provide therapeutic efficacy. While it is known that tumor presence can alter the immune system, very few studies have explored this impact on nanoparticle circulation. In this report, we demonstrate how the presence of a tumor can change the local and global immune system, which dramatically increases particle clearance. We found that tumor presence significantly increased clearance of PRINT hydrogel nanoparticles from the circulation, resulting in increased accumulation in the liver and spleen, due to an increase in M2-like macrophages. Our findings highlight the need to better understand interactions between immune status and nanoparticle clearance, and suggest that further consideration of immune function is required for success in preclinical and clinical nanoparticle studies.

Purpose We evaluated the role of a poly(ethylene glycol) (PEG) surface coating to increase residence times and alter the cellular fate of nano- and microparticles delivered to the lung. Methods Three sizes of PRINT hydrogel particles (80×320 nm, 1.5 and 6 um donuts) with and without a surface PEG coating were instilled in the airways of C57/b6 mice. At time points of 1, 7, and 28 days, BALF and whole lungs were evaluated for the inflammatory cytokine Il-6 and chemokine MIP-2, histopathology, cellular populations of macrophages, dendritic cells (DCs), and granulocytes, and particulate uptake within these cells through flow cytometry, ELISAs, and fluorescent imaging. Results Particles of all sizes and surface chemistries were readily observed in the lung with minimal inflammatory response at all time points. Surface modification with PEGylation was found to significantly increase lung residence times and homogeneous lung distribution, delaying macrophage clearance of all sizes, with the largest increase in residence time observed for 80×320 nm particles. Additionally, it was observed that DCs were recruited to the airway following administration of unPEGylated particles and preferentially associated with these particles. Conclusions Pulmonary drug delivery vehicles designed with a PEG surface coating can be used to delay particle uptake and promote cell-specific targeting of therapeutics.

Cisplatin is a cytotoxic drug used as a first-line therapy for a wide variety of cancers. However, significant renal and neurological toxicities limits it clinical use. It has been documented that drug toxicities can be mitigated through nanoparticle formulation, while simultaneously increasing tumor accumulation through the enhanced permeation and retention effect. Circulation persistence is a key characteristic for exploiting this effect, and to that end we have developed long-circulating, PEGylated, polymeric hydrogels using the Particle Replication In Non-wetting Templates (PRINT®) platform and complexed cisplatin into the particles (PRINT-Platin). Sustained release was demonstrated, and drug loading correlated to surface PEG density. A PEG Mushroom conformation showed the best compromise between particle pharmacokinetic (PK) parameters and drug loading (16 wt %). While the PK profile of PEG Brush was superior, the loading was poor (2 wt %). Conversely, the drug loading in non-PEGylated particles was better (20 wt %), but the PK was not desirable. We also showed comparable cytotoxicity to cisplatin in several cancer cell lines (non-small cell lung, A549; ovarian, SKOV-3; breast, MDA-MB-468) and a higher MTD in mice (10 mg/kg versus 5 mg/kg). The pharmacokinetic profiles of drug in plasma, tumor, and kidney indicate improved exposure in the blood and tumor accumulation, with concurrent renal protection, when cisplatin was formulated in a nanoparticle. PK parameters were markedly improved: a 16.4-times higher area-under-the-curve (AUC), a reduction in clearance (CL) by a factor of 11.2, and a 4.20-times increase in the volume of distribution (Vd). Additionally, non-small cell lung and ovarian tumor AUC was at least twice that of cisplatin in both models. These findings suggest the potential for PRINT-Platin to improve efficacy and reduce toxicity compared to current cisplatin therapies.

Platinum nanoparticles (PtNPs) have shown promise as diagnostic and therapeutic agents due to their unique physiochemical properties. However, critical parameters, such as toxicity and accumulation at both desired and other tissues, remain a significant concern in the clinical translation of these nanomaterials. Here, we examine the cytotoxicity, biodistribution, and effect on clearance organ function of an intravenously administered polyethylene glycol (PEG) -ylated PtNP construct. We synthesized hydrophobic PtNPs and assembled them into aqueous micelles with the lipid-polymer conjugate 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG (PtNP: DSPE-PEG, ~70 nm). This construct was well tolerated in mice receiving up to 15 mg platinum per kg body weight with no observed loss in weight, plasma chemistry within normal healthy ranges, and normal histopathology of organs after three weeks. Platinum quantification studies (inductively-coupled plasma mass spectroscopy (ICP-MS)) were also performed to assess biodistribution of PtNPs. The findings of this study are consistent with the in vivo accumulation of metal nanomaterials and further highlight the need to address clearance when designing nanomaterials for medical applications.