Folate-targeted Paclitaxel-conjugated Polymeric Micelles Inhibits Pulmonary Metastatic Hepatoma In Experimental Murine H22Metastasis Models

The folate receptor (FR) is over-expressed in many types of human cancers. It canmediate endocytosis of folic acid (FA) or FA-carrying drugs. Based on this specific interactionbetween FA and FR, folic acid and paclitaxel (PTX) containing micelles (FA-M(PTX)) wereprepared by co-assembling an FA-polymer conjugate, poly(ethylene glycol)-b-poly(L-lactide-co-2,2-dihydroxylmethyl-propylene carbonate/FA)[PEG-b-P(LA-co-DHP/FA), FAcontent1.4wt%] and a PTX-polymer conjugate, poly(ethylene glycol)-b-poly(L-lactide-co-2-methyl-2-carboxyl-propylene carbonate/PTX)[PEG-b-P(LA-co-DCC/PTX), PTXcontent25wt%] with a mass ratio of1:9. Anti-tumor activity of FA-M(PTX) on H22cancerswere evaluated on BALB/c mice models by pulmonary burden measurements, survival timeanalysis, flow cytometry assay, immunohistochemistry (IHC) and histopathology. The resultsshowed that FA-M(PTX) exhibits significant anti-tumor effects in vivo compared to pure PTXand simple PTX-polymer micelles. This enhanced efficacy of FA-M(PTX) is ascribed to thetargeting effect of FA moieties in the micelles. Therefore, conjugation of both PTX and FAonto the hydrophobic segments of the block copolymer and co-assembling of thePTX-polymer and FA-polymer conjugates are successful strategies to construct cancer celltargeting drug delivery systems.In this research, folic acid (FA) was chosen as the targeting moiety, and it is attached to acarrier polymer via an ester linkage. Based on our previous work, paclitaxel (PTX) isconjugated to another carrier polymer. These two carrier polymers are co-assembled intocomposite micelles which are expected to have many properties including water solubility,prolonged circulation, biodegradability, folate-receptor targeting and rapid cell uptake. Toexamine the anti-tumor efficacy of these composite micelles, murine H22cancer xenograftnude mice models were constructed and a series of evaluation was performed such as tumorgrowth inhibition rate, survival time analysis, immunohistochemistry (IHC) andhistopathology. The results showed that FA-M(PTX) exhibits significant anti-tumor efficacy in vivo compared to pure PTX and simple PTX-polymer micelles. This enhanced efficacy ofFA-M(PTX) is ascribed to the targeting effect of FA moieties in the micelles.Results and conclusions of this study:1. In vitro1.1The cell viability of H22incubated with PTX, M(PTX), and FA-M(PTX) for12hours,24hours, and48hours are depicted in Figure1as a function of the equivalent PTXconcentration.1.2The time-dependent apoptosis of H22cells induced by the drugs was observed. As shownin Figure2, the apoptosis of the drug groups were evidently higher when compared with thecontrol at24hours and72hours. The mean apoptosis rate of PTX, M(PTX), and FA-M(PTX)was16.8%,8.8%, and11%at24hours, while it was up to39.4%,34.9%, and37.7%at72hours, respectively.1.3The three cell lines were exposed to0.1%FA-f-micelles and FA-mixed micelles for2hours,respectively, and a quantitative analysis by FCM was performed. It is presumed and confirmed in this studythat the cellular uptake of FA-mixed micelles was increased in HeLa and H22cells with overexpressed FRwhen compared to nontumorigenic BEAS-2B cells. As shown in Figure3, the obvious preferentialuptake of FA-mixed micelles was shown in cancer cells (HeLa,62.1%; H22,55.4%) compared withBEAS-2B cells (37.6%). Furthermore, an increased uptake of FA-f-micelles by HeLa (41.8%) and H22(40.1%) was observed when compared with BEAS-2B (36.5%). Statistically, HeLa and H22revealed theenhanced cellular uptake of FA-mixed micelles compared with BEAS-2B (P<0.001and P=0.017,respectively). Moreover, the uptake of FA-mixed micelles by the two cancer cells was significantlyincreased compared with that of FA-f-micelles (P<0.001). In addition, HeLa showed the preferential uptakeof FA-mixed-micelles compared with H22(P=0.036), which implicated FA–FR mediated endocytosis.2. In vivo2.1The density of the metastatic colonies was visibly decreased in the drug groups comparedwith control. As shown in Figure4, the ratio of wet lung weight to body weight in the druggroups were statistically decreased as an index of metastasis burden when compared with control (P<0.05), and the ratio varied in an almost dose-related manner.2.2The microscopic tumor metastasis colonies detected by HE decreased in the drug groupsin the order of PTX <M(PTX)<FA-M(PTX) when the equivalent dose of PTX is15mg/kgor30mg/kg; furthermore, a dose-dependent therapeutic effect was indicated. In addition, inthe groups of15mg/kg, there were no significant difference between M(PTX) andFA-M(PTX), while the grey value of FA-M(PTX) as MMP-9was evidently higher than thatof the M(PTX) group. In conclusion, the expression of MMP-2and MMP-9weredownregulated in the drug groups, and FA-M(PTX) demonstrated the most efficaciousinhibition of the expression of MMP-2and MMP-9, which implied that the FA-M(PTX) wassuperior than PTX and M(PTX) for the treatment of pulmonary metastatic hepatoma in vivo,given all the conditions in this study.2.3The Kaplan–Meier survival curve is shown in Figure6. The median/mean survival time ofthe groups was compared using the log-rank test. The mice in the drug groups showed aprolonged survival time compared with the control with equivalent doses of PTX of15mg/kgas well as30mg/kg. The FA-M(PTX) was significantly more effective than pure PTX and M(PTX)on H22pulmonary metastasis models.