Supplementary MaterialsSupplementary Document. effectiveness of iCluster than its control organizations, demonstrating that overcoming these delivery obstacles may be accomplished by innovative nanoparticle style. and and and images, 50 nm.) (and and and and and and = 3). * 0.05, *** 0.001. Next, cell internalization of both nanoparticles was studied by flow cytometry (FACS) and ICP-MS, respectively. In FACS analysis, MCSs were treated with iClusterFlu, ClusterFlu, or PAMAMFlu at pH 6.8 for 4 h and 24 h. Compared with ClusterFlu treatment, the population of positive cells treated with iClusterFlu was 1.7-fold higher and 3-fold higher at 4 h ( 0.05) and 24 h ( 0.001), Phloridzin cell signaling respectively (Fig. 2and shows that iCluster/Pt treatment resulted in significantly higher intracellular accumulation of Pt than Cluster/Pt treatment (2.6-fold, 0.001). Moreover, DNA of MCS cells were isolated after a 24-h incubation, and the amount of Pt binding to these DNA in MCS cells receiving iCluster/Pt treatment was Phloridzin cell signaling significantly higher than that receiving Cluster/Pt treatment (3.6-fold, 0.001, Fig. 2 0.001, Fig. 2and 0.001, versus Cluster/Pt treatment). The average weight of the tumor mass excised at the end of treatment also demonstrated the same trend (and 0.001. ( 0.05, ** 0.01. (and and 0.01, *** 0.001. Data are presented as mean SD = 3 for and = 5 for and 0.01 for 12 h, and 0.05 for 24 h) and at least 7-fold higher than free cisplatin and PAMAM/Pt (Fig. 3 0.05 for 12 h and 0.001 for 24 h; Fig. 3 0.05) and 24 h (3-fold, 0.001) (Fig. 3 0.001). Of note, the significance between iCluster/Pt and Cluster/Pt appeared as early as day 9 postinjection. No obvious body weight loss was observed for the nanoparticle formulations (= 5). ** 0.05, *** 0.001. (= 10). Mice were treated at a platinum dose of 3 mg/kg via i.v. administration on days 10, 15, and 20 after tumor inoculation. To further extend the applicability of our strategy to combat metastatic cancer, we established a highly invasive and metastatic 4T1 orthotopic tumor model, which is known to be more aggressive and more refractory to chemotherapy than a s.c. tumor model (43). The mice were treated with varying Pt-containing formulations, and their survival curves were recorded. Compared with PBS and blank iCluster control groups, all other treatments showed improved median survival time. In particular, the iCluster/Pt treatment improved survival time by 74.2%, with significantly longer time to end point than that of Cluster/Pt (Fig. Phloridzin cell signaling 5and em SI Appendix /em , Table S4). The comparison of intratumoral microdistribution of RhBiClusterFlu and RhBClusterFlu in A549R and 4T1 tumor cells also proven that iCluster demonstrated far better tumor penetration than Cluster for their pHe-activated PAMAM launch in the tumor site ( em SI Appendix /em , Figs. S20 and S21), once again indicating that the improved antitumor activities of iCluster are highly associated with enhanced tumor penetration. Discussion Despite the fact that nanoparticle-based therapeutics are amenable to preferential accumulation in solid tumors by taking advantage of the EPR effect, they encounter a series of sequential biological barriers upon i.v. administration, which severely impede the achievement of optimal therapeutic outcomes. To adequately address these barriers and achieve effective Phloridzin cell signaling therapy, nanoparticles must be rationally designed to overcome substantial interstitial transport hindrance brought about by their inherently large sizes to realize deep and uniform tumor penetration (7). In this study, our iCluster system enables its basic physicochemical properties to adaptively change in response to the endogenous stimuli of the tumor microenvironment to accomplish improved therapeutic efficacy by successively increasing blood circulation and tumor vascular extravasation, improving tumor penetration, facilitating cell internalization, and accelerating intracellular drug release. Our results demonstrate that the decisive step for the effectiveness of iCluster is its robust tumor penetration achieved through pHe-triggered shattering of small PAMAM dendrimers at tumor sites (Figs. 3 and ?and4).4). It has been validated that the penetration of nanoparticles in tumor space relies heavily on particle size, with the consensus that smaller particles have improved tissue penetration (26, 33, 44). Such progress has recently inspired interest in developing size-shrinkable anticancer drug Phloridzin cell signaling delivery systems (15, 34, 45, 46). Compared with previous studies, our strategy has several unique features. First, previous delivery systems simply Kcnj12 focused on size-shrinkage medicated tumor penetration, whereas our system is devised to systematically overcome a series of barriers including tumor penetration. Attaining this objective is certainly essential because these obstacles are interconnected vitally, and simply conquering one individual hurdle is not sufficient to produce correct therapeutic final results (42, 47). Second, the stimuli which were utilized to cause size shrinkage had been either by enzyme or UV light previously, whose applicability, to a certain degree, would be limited to just a subset.