Integration of Nanoassembly Functions for an Effective Delivery Cascade for Cancer Drugs

A "cluster-bomb"-like lipid–dendrimer nanoassembly synergizes the functions of its components and thereby efficiently accomplishes the drug delivery cascade for high efficacy in treating cancer. The nanoassembly successfully circulates in the blood and accumulates in the tumor. Once in the tumor, it releases small dendrimers that act like "bomblets", enabling tumor penetration, cell internalization, and drug release. Cancer nanomedicine is regarded as one of the few strategies that may revolutionize cancer treatments.1 Several nanomedicines such as DOXIL are in clinical use, and many more are under clinical trials.2 A cancer nanomedicine delivering active drugs to the cytoplasm of cancer cells in a solid tumor must go through a cascade of five steps, i.e., circulation in the blood compartments (C), accumulation in the tumor via the enhanced permeability and retention (EPR) effect (A),3 subsequent p–enetration deep into the tumor tissue (P), internalization by tumor cells (I), and finally intracellular drug release (R)—abbreviated as the CAPIR cascade (Figure 1a). Thus, a nanosystem would produce high therapeutic efficacy and good prognosis only if it efficiently accomplishes the full CAPIR cascade.4 This may account for the difficulty of current nanomedicines5—they fail to or only marginally improve the overall survival of patients even though they deliver more anticancer agents to tumor tissues than free drugs.6 For example, DOXIL effectively accumulates in the tumor tissues but cannot penetrate in the tumor, resulting in low therapeutic efficacy.7 In our approach of integrating the functions of various materials, we made a cluster-bomb-like nanocarrier that synergizes the various functions of its components, thereby enabling it to complete the CAPIR cascade and achieve high therapeutic efficacy. The utmost challenge in the design of a CAPIR-capable nanocarrier is the integration of the necessary functions into a single system since the required functions may be opposite in different CAPIR steps. For example, PEGylation8 (PEG = poly(ethylene glycol)) and other designs9 enable nanocarriers to be stealthy and thus long circulating, but these strategies substantially hinder their ability for cellular internalization.10 Introducing targeting ligands11 or cationic charges to nanocarriers promotes their cellular uptake,12 but these techniques may greatly shorten the carrier's blood circulation and impede tumor penetration.13 Nanoparticles of 100 nm have longer blood circulation times and better tumor accumulation than smaller particles,14 but they are too large to diffuse into tumor tissues composed of tightly packed cells in a dense extracellular matrix.7, 15 Small nanocarriers can penetrate tumors,4, 16 but very small particles have short-lived blood circulation.17 Many stimuli-responsive18 or recent multistage nanosystems19 have tried to accommodate the required opposing functions with various strategies: PEGylation and dePEGylation,20 a large-to-small size transition,21 or a negative-to-positive charge transition,22 all of which are triggered by tumor microenvironment. None of these previously reported nanosystems, however, could simultaneously integrate all of the necessary opposing functions into one system. We accommodated the opposing functions using a dendrimer–lipid nanoassembly that undergoes the three needed transitions for the CAPIR cascade similar to the way a cluster bomb undergoes two stage-actions; the nanoassembly accomplishes the missions of blood circulation and tumor accumulation (the CA steps of CAPIR), while its small nanocarrier components act as "bomblets" accomplishing the missions of tumor penetration and cell internalization as well as drug release (the PIR steps of CAPIR) (Figure 1b). Lipids are most clinically used for formulating drug delivery nanocarriers23 and recently also used as coatings for nanostructures.24 Hence a fusogenic phospholipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) was chosen to coat the dendrimers, but it was expected to peel off by fusion once inside tumor tissue. A PEGylated lipid DSPE-PEG (DSPE = 1,2-distearoyl-sn-glycero-3-phosphoethanolamine) and cholesterol (Figure 1c) were added to make the nanoassembly stealthy and stable in the blood. Dendrimers were chosen as the "bomblets" because of their monodispersity, uniform nanoscale size, and ability to carry anticancer drugs.25 A sixth-generation nontoxic degradable polyaminoester dendrimer with a diameter of 5 nm was synthesized (Figure 1c)26 with pH-dependent 2-(N,N-diethylamino)ethyl termini. The zeta-potential was 2.2 mV at pH 7.4 and 7.0 mV at pH 6.5 in 0.2 m phosphate buffered saline (PBS) solution (Figure S1, Supporting Information (SI)). Thus, the dendrimer can be quickly internalized at acidic pH (SI: Figure S2), thereby shipping the drugs into the cytosol and circumventing the cell's multidrug resistance (SI: Figure S3, S4). Furthermore, the dendrimer's core is hydrophobic at neutral pH, but it becomes water-soluble at a pH of ~6 because it contains many tertiary amines in its backbone. Thus, hydrophobic drugs could be encapsulated inside the dendrimer at neutral pH and released once at lower pH.27 The self-assembly of the dendrimer with DOPE lipid, cholesterol, and DSPE-PEG (abbreviated as D, L, C, and PEG, respectively, in the nanoassembly label: DLC-PEG) was fine-tuned in terms of the size, zeta-potential, and stability (SI: Figure S5). The optimum PEGylated DLC-PEG nanoassembly was obtained at a D:L:C:PEG molar ratio of 1:60:60:1.5 with a size of 30 ± 2 nm (polydispersity index, PDI = 0.163) and zeta-potential of –9.1 ± 0.5 mV. The structure of the DLC-PEG nanoassembly was probed by regular and cryogenic transmission electron microscopy (TEM and cryo-TEM, respectively). As illustrated in Figure 1c (and in the SI: Figure S6), both regular TEM (with negative staining using hydrophilic uranyl acetate) and cryo-TEM images showed a dim ring surrounding a bright core, indicating a hydrophobic core surrounded by a hydrophilic layer. Assuming no deformation occurred when the dendrimers packed together, it was therefore estimated that the DLC-PEG nanoassembly (30 nm in diameter) roughly contained 27 dendrimers (5 nm in diameter) aggregated together and coated with a lipid monolayer. The nanoassembly structure was further probed using a fluorescence quenching approach. A fluorescent FITC-tethered dendrimer (FITC = fluorescein isothiocyanate; FITCD = tethered dendrimer; SI: Figure S7a,b) was used to fabricate the labeled nanoassembly FITCDLC-PEG. Gold nanoparticles (AuNPs, 4 nm, see SI: Figure S7c) are known to quench the FITC fluorescence and are too big to diffuse through a lipid layer. Thus, the fluorescence of free FITCdendrimer in solution was gradually quenched upon addition of AuNPs (SI: Figure S7d,e). However, adding AuNPs to the FITCDLC-PEG nanoassembly solution only slightly reduced the fluorescence intensity (SI: Figure S7f). This suggests that most of the FITC were inaccessible to the 4-nm AuNPs, further confirming that most of the FITCD (82%) were encapsulated by a lipid layer within the nanoassembly structure. An anticancer drug doxorubicin (DOX) was loaded into the DLC-PEG nanoassembly. The resulting DLC-PEG/DOX had a slightly larger diameter, 45 ± 5 nm, and a DOX-content of 9 ± 2 wt%. The DLC-PEG/DOX could be lyophilized with trehalose—a known lyoprotectant reagent (DLC-PEG:trehalose w/w ratio = 1/12)—and easily redispersed in aqueous solution with almost no effect on its size. The DOX-loaded DLC-PEG released DOX more slowly than the unbound dendrimers and the dendrimer-free assembly of LC-PEG, indicating that DOX was primarily encapsulated in the dendrimers (SI: Figure S8). Although the release profile was still not ideal, changing the dendrimer to one that can hold DOX more tightly has the potential to greatly suppress premature release. The cellular uptake of DLC-PEG into cells of the SKOV-3 cell line (human ovarian cancer cells) was observed using confocal microscopy. The FITCD (green signal) and some DOPE labeled with rhodamine B (RHoBL, red signal) were employed to form the dual-labeled nanoassembly FITCDRHoBLC-PEG (yellow spots). After FITCDRHoBLC-PEG was incubated with cells for 4 h, the free dendrimer, free lipid, and the nanoassembly were found attached to the cell membrane (Figure 2a). After 12 h incubation, the lipids were still on the cell membrane, but the dendrimers were found inside the cells, suggesting that the nanoassembly dissociated and released the dendrimers. Furthermore, the DOX in the DLC-PEG nanoassembly was in the cytoplasm after 12 h incubation (SI: Figure S9). Notably, dendrimer/DOX (red signal), which was delivered by the nanoassembly, was not localized in the lysosomes (green signal) (Figure 2b). In contrast, the cultured free dendrimers were found in lysosomes (SI: Figure S10). The effects of temperature and endocytosis pathway inhibitors (chlorpromazine, filipin III, and wortmannin) on the cellular uptake of the nanoassembly were probed by flow cytometry (Figure 2c). Cellular uptake was suppressed to half at 4 °C, suggesting that the internalization was mainly energy-dependent. The presence of chlorpromazine, filipin III, or wortmannin had almost no effect on the cellular uptake of the nanoassembly. A similar result was obtained when adding cytochalasin D, a commonly used inhibitor of actin-polymerization for phagocytosis (SI: Figure S11). The results indicate that cellular uptake of the nanoassembly was not through the common endocytosis, macropinocytosis, or phagocytosis pathways. We used DOPE for its fusion capability to induce the disassembly of DLC-PEG after interacting with tumor cells and probed it by a fluorescence-resonance energy-transfer (FRET) approach.28 DOPE lipids were separately labeled with a pair of FRET dyes, RHoB and NBD (RHoBDOPE and NBDDOPE), and mixed with DOPE at a DOPE:RHoBDOPE:NBDDOPE molar ratio of 94:1:5 to form the nanoassembly with dual-labeled lipid, DRHoB/NBDLC-PEG (Figure 2d). If the lipid layer fuses with the cell membrane, the RHoBDOPE/NBDDOPE lipids will migrate to the cell membrane and separate, inhibiting their FRET. A FRET efficiency index, R, can be calculated from the intensity ratio of the RHoBDOPE fluorescence at 585 nm to the NBDDOPE fluorescence at 525 nm when excited at 450 nm.29 Upon excitation at 450 nm, the intact DRHoB/NBDLC-PEG nanoassembly had a strong FRET-fluorescence peak at 585 nm and a weak peak at 525 nm with an R value of 3.4 (R = 178.6/51.9). In the solubilized control solution, the addition of 0.24 vol% Triton X-100 led to the nanoassembly dissolving and dissembling, completely eliminating FRET. Interestingly, the cells treated with the DRHoB/NBDLC-PEG nanoassembly had very strong florescence at 525 nm but a very weak FRET peak with an R value of 0.39 (R = 7.79/19.81; Figure 2d, right), indicating that many of the nanoassembly lipids were in the cell membrane but that they had separated. This finding is in agreement with the confocal microscopy observations and with the flow cytometry results (Figure 2a,c), in which the nanoassembly fused with the cell membrane and released the dendrimers directly into the cytoplasm. The (lipid layer)–(cell membrane) fusion was also designed to strip off the lipid layer from the DLC-PEG nanoassembly to extracellularly release the dendrimers for penetration into the tumor. Since tumor tissues are tightly packed with cells in a dense extracellular matrix, this fusion would be more feasible than that in blood. To test the extracellular dendrimer release, we incubated FITCDLC-PEG with high-density SKOV-3 cells (2.6 × 104 cells/cm2) at 37 °C and collected the extracellular culture medium. The FITCD fluorescence in the medium was measured, and the nanoassembly integrity was probed again using the AuNP quenching method (Figure 2e and SI: Figure S12). After the nanoassembly was incubated with the cells for 5 min, some FITCD fluorescence became quenchable by the AuNPs, and the peak FITC emission shifted from 520 to 530 nm, which is typical for FITC in a hydrophilic environment. This phenomenon became much more pronounced after longer incubation. After 45 min of incubation, the FITCD fluorescence was completely quenched after adding 100 μL of the AuNP solution, which is very similar to free FITCD, indicating that the dendrimers in the extracellular solution were in an aqueous environment. Therefore, it can be concluded that the lipid layer of the nanoassembly could fuse with the cell membrane, releasing the dendrimers either into the cell cytosol or the extracellular medium as we had intended (Figure 1b). The cytotoxicity of the DOX loaded in DLC-PEG (DLC-PEG/DOX) to five cancer cell lines was slightly lower than that of free DOX (SI: Figure S13), suggesting that DOX was not taken into lysosomes but released into the cytosol to exert its efficacy. The blank, DLC-PEG without any DOX, had no cytotoxicity. Importantly, the cytosolic dendrimer/DOX remained in the cells and did not undergo exocytosis, whereas intracellular free DOX was quickly excluded from the cells within 10 h (SI: Figure S14), indicating that the dendrimer/DOX indeed could overcome the membrane-associated drug resistance. Several tumor models were used to evaluate the nanoassembly's ability to accomplish the CAPIR process. The in-vivo stealthy property of DLC-PEG was first compared with a standard long-circulating nanocarrier of similar size, polycaprolactone-block-PEG (PCL-PEG; sometimes subscripts are included to indicate the size of each block) micelles. Both the dendrimers in the nanoassembly and the PCL-PEG (the PCL end) were conjugated with FITC for non-leaching labeling. A hydrophobic fluorescence probe, DiR, was loaded as a model drug into FITCDLC-PEG and FITCPCL-PEG for tracing because the fluorescence wavelengths of DOX and FITC partially overlap. DiR- and DOX-loaded DLC-PEG had similar sizes, blood circulation times, biodistributions, and extraction efficiencies (SI: Figure S15). DLC-PEG/DiR was found to circulate in the blood in a similar manner as the well-known long-circulating PCL-PEG/DiR micelles, suggesting that the nanoassembly was stable in blood and indeed had good stealthiness (Figure 3a). Their biodistribution profiles in the liver, spleen, and kidneys were not significantly different at 16 h post-injection, but significantly different in the tumor, lung, and blood (SI: Figure S16). DLC-PEG/DiR accumulated more in the tumor cells than PCL-PEG/DiR in terms of the overall fluorescence intensity of either FITC or the DiR in the tumor cells. The amount of FITCD accumulated in tumor cells was 1.7 (statistical P-value of P = 0.038) times that of FITC-labeled PCL-PEG; similarly, the amount of DiR-loaded DLC-PEG accumulated in the tumor was 1.5 times that of the DiR-loaded PCL-PEG (Figure 3b and SI: Figure S17). The dual-labeled FITCDRHoBLC-PEG nanoassembly was loaded with DiR (FITCDRHoBLC-PEG/DiR) and used to observe the intratumoral distribution, nanoassembly dissociation, and drug release (Figure 3c). The FITCD (green signal) was separate from the RHoBDOPE (red signal), suggesting dissociation of the dendrimers and lipid layer in the tumor. The green signal of the FITCD and red signal of DiR in Figure 3d mostly overlapped as yellow spots after the nanoassembly extravasated from the blood vessel into the tumor (Figure 3, magnified area 1), and they mostly overlapped during deep penetration through the tumor tissue (Figure 3, magnified area 2), indicating that the dendrimers retained DiR well. Furthermore, the dendrimers distributed throughout the tumor (Figure 3d, large view). We further examined the tumor accumulation and penetration of DOX delivered by DLC-PEG in BCAP-37 and MCF-7 tumor-bearing mice and compared them with similar-sized PCL2k-PEG2k/DOX nanoparticles (40 nm in diameter). The DLC-PEG delivered 1.7 (P < 0.05) times as much DOX as PCL2k-PEG2k (Figure 4a), similar to the DiR result (1.9-fold, P < 0.05; SI: Figure S19). The confocal image of a tumor slice clearly showed that DOX was much more homogeneously distributed in the tumor, whereas the DOX delivered by PCL2k-PEG2k was mostly retained in the invasive edge (Figure 4b,c). The calculated DOX intensity (IDOX) inside the tumor treated with DLC-PEG/DOX was about 10 times more than that treated with PCL2k-PEG2k/DOX (Figure 4b). Similar results were found in the MCF-7 tumor model (SI: Figure S20). These results further prove that the nanoassembly DLC-PEG could release the dendrimers once in the tumor, and the small dendrimers penetrated more deeply into the whole tumor tissue. The in-vivo therapeutic efficacy of DLC-PEG/DOX was compared with the micellar PCL-PEG/DOX using subcutaneous tumor-bearing mice of a well-established cancer cell line pair, drug-sensitive/resistant MCF-7 and MCF-7/ADR breast cancer cells (Figure 5a; SI: Figure S21 and S22). The DLC-PEG/DOX lyophilized with trehalose was redissolved in pure water to appropriate doses. Thus, all other treatments were also administered in the same trehalose solution (60 mg/mL), and the trehalose solution was used as a control. Clearly, DLC-PEG/DOX and free DOX inhibited the growth of drug-sensitive MCF-7 tumors completely, much more efficiently than PCL2k-PEG2k/DOX (P < 0.005). On day 19, DLC-PEG/DOX had an 85% tumor inhibition rate in terms of tumor weight, much higher than PCL2k-PEG2k/DOX (40%). Hematoxylin–eosin (H&E) staining (Figure 5b) showed that tumors treated with trehalose or the blank nanoassembly consisted of typical, tightly packed tumor cells, whereas those treated with DOX or DLC-PEG/DOX exhibited extensive vacuolization. TUNEL staining (TdT-mediated dUTP nick end labeling) confirmed that most MCF-7 tumor cells treated with DLC-PEG/DOX or DOX were TUNEL-positive (brown staining) and scored as apoptotic, but fewer apoptotic cells were found in tumors treated with PCL2k-PEG2k/DOX. When used for treatment of drug-resistant MCF-7/ADR tumor-bearing mice, free DOX or PCL2k-PEG2k/DOX could not inhibit the tumor growth anymore (P > 0.05 compared with the trehalose group), whereas DLC-PEG/DOX almost completely stopped the tumor growth, and the difference was more significant (P < 0.005) after day 17 (Figure 5a). On day 19, DLC-PEG/DOX had a tumor inhibition rate of 63%, but it was only 32% for DOX and 21% for PCL2k-PEG2k/DOX, which were similar to the control. H&E and TUNEL staining (Figure 5b) showed that the MCF-7/ADR tumors treated with DLC-PEG/DOX consisted mostly of apoptotic cells, but such cells were rare in those treated with PCL2k-PEG2k/DOX or DOX. Coupled with the tumor penetration results as discussed above, the difference in therapeutic efficacy suggests that DOX could not get into the drug-resistant cells and had low efficacy. PCL-PEG micelles could circumvent the membrane-associated drug resistance via endocytosis and take DOX into drug-resistant cells,30 but it could not penetrate into the tumor tissue to reach cells away from the tumor blood vessels. In contrast, DLC-PEG/DOX released the drug-loaded dendrimers, which further penetrated deep in tumor tissues. Importantly, the dendrimers became positively charged in the acidic tumor extracellular fluids31 and were efficiently internalized via endocytosis (SI: Figure S3, S4), circumventing the drug resistance and leading to significantly enhanced therapeutic efficacy, as sketched in Figure 1b. During the treatments, DOX caused a significant loss of mouse body weight and histological changes in the cardiomyocytes; in contrast, DLC-PEG/DOX treatment did not cause this weight loss, and the treated mice exhibited no differences in cardiomyocyte structures compared to trehalose- and blank-nanoassembly-treated mice (SI: Figure S23). This study was funded by the National Natural Science Foundation Key Program (51390481 and 21090352), the National Basic Research Program (2014CB931900), the National Fund for Distinguished Young Scholars (50888001), and the Doctoral Fund of Ministry of Education (20110101130007) of China, and the US Department of Defense (BC090502). Cryo-TEM characterizations were carried out in the Characterization Facility, University of Minnesota, which receives partial support from the NSF through the MRSEC program. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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