Filomicelles Deliver a Chemo-Differentiation Combination of Paclitaxel and Retinoic Acid That Durably Represses Carcinomas in Liver to Prolong Survival

Abstract

Drug resistance and relapse is common in cancer treatments with chemotherapeutics, and while drug combinations with naturally occurring, differentiation-inducing retinoic acid (RA) provide remission-free cures for one type of liquid tumor, solid tumors present major problems for delivery. Here, inspired by filoviruses that can be microns in length, flexible filomicelles that self-assemble from an amphiphilic block copolymer (PEG-PCL) are shown to effectively deliver RA and paclitaxel (TAX) to several solid tumor models, particularly in the liver. These hydrophobic compounds synergistically load into the cores of the elongated micelles, and the coloaded micelles prove most effective at causing cell death, ploidy, and durable regression of tumors compared to free drugs or to separately loaded drugs. RA-TAX filomicelles also reduce mortality of human lung or liver derived cancers engrafted at liver, intraperitoneal, and subcutaneous sites in immunodeficient mice. In vitro studies show that the dual drug micelles effectively suppress proliferation while upregulating a generic differentiation marker. The results highlight the potency of dual-loaded filomicelles in killing cancer cells or else driving their differentiation away from growth.

Figure 1.

(A) Schematic depicting effect of RA, TAX, and RA-TAX on cells after filomicelles release drugs. Only RA-TAX combination leads to durable effects. (B) Cells treated with RA consistently increase in number, consistent with RA not killing cells, just differentiating them. TAX treated cells decline in number initially (similar to initial tumor shrinkage), but cell death plateaus after a week when proliferating cell numbers overtake dying ones. RA and TAX treated cells, on the other hand, consistently decrease in number, indicating a more durable treatment. (C) Quantification of DNA content and cell size after drug treatment. DNA content increases for TAX and RA-TAX due to incomplete cell division; it decreases for RA due to a lower number of proliferating cells (and hence lower DNA replication before division). DNA content for cells treated with single drugs returns to normal after about 6 days (about 3 days after treatment), indicating the transient nature of single drugs, whereas DNA content for RA-TAX decreases much more slowly. Similarly, cell size for single drugs returns to normal within 3 days, indicating the transient nature of single drugs. However, they remain consistently high for combination treatment. (D) Delivery systems with two payloads can be coloaded onto the same micelle or on separate filomicelles which are then mixed. At high concentrations, both are equally effective. However, at therapeutic concentrations (around IC50), coloaded filomicelles are 4 times more potent than separately loaded filomicelles (inset bar graph). (E) Most wells treated with TAX relapse (92%), the rate is much lower for RA-TAX treated wells (15%) 22 days post-treatment, indicating that 85% of wells exposed to RA-TAX die, which illustrates a much more durable treatment than the single drug. Loading TAX onto filomicelles does not alter relapse (89% of TAX treated wells experienced relapse), highlighting again the limitation of a single drug. The efficacy of RA-TAX was greatly improved via loading onto filomicelles, with no relapses in cells observed after 30 days.

Figure 2.

(A) td-Tomato images of subcutaneous tumors on untreated, TAX and RA-TAX treated mice at day 52. RA-TAX treated mice had the smallest tumors and td-Tomato intensity. (B) Mice bearing A549 subcutaneous tumors were injected 4 times with single drug (TAX, black points) or combination (RA-TAX, green points) loaded into filomicelles. RA-TAX combination produced the most tumor shrinkage (30%) which remained shrunk for 20 days and grew slowly after that.

Figure 3.

(A) td-Tomato images of liver and subcutaneous tumors on untreated, RA, TAX, and RA-TAX treated mice at day 55. RA-TAX treated mice had the lowest td-Tomato intensity in both cases. (B) RA-TAX filomicelles were efficacious with orthotopic A549 liver tumors, shrinking and arresting the growth of tumors, while single drugs failed to do both. While TAX controlled the growth of tumors, those treated with RA continue to grow. (C) RA-TAX filomicelles shrunk subcutaneous A549 tumors by ~50%. Single drug controls arrest the growth of tumors 30 days from the start of the treatment. During the same time frame, untreated tumors more than double in size. (D) Kaplan−Meier curve showing significant prolonged survival of RA-TAX treated mice A549 liver and subcutaneous tumors.

Figure 4.

(A) RA-TAX suppressed the growth of HepG2 subcutaneous tumors in vivo. Mice treated with RA-TAX filomicelles experienced 30% growth in tumors, while untreated tumors more than tripled in size during the same time frame. Free RA-TAX produced minimal retardation in tumor growth. (B) Orthotopic liver tumors established in vivo from HepG2 cells. RA-TAX treated tumors are 65% smaller than untreated tumors. (C) Quantification of liver tumor size from (B) and antihuman stain in adjacent liver lobe to identify number of HepG2 cells migrating. Quantification of cells staining positive is depicted in the bar graph. (D) Kaplan−Meier curve showing significant prolonged survival of RA-TAX treated mice with HepG2 liver tumors.

Figure 5.

(A) Bar graph representation of the fold change in IC50 with RA-drug combination. Combination of 5-fluorouracil shows negligible change in IC50, and oxaliplatin-RA showed a 1.6-fold change in IC50, signifying some synergy. However, combination of RA and Paclitaxel had a 2.5-fold reduced IC50 than parent chemotherapeutic (Paclitaxel), signifying maximum synergy among the three combinations and the best candidates for further experiments. (B) Molar ratio dictates improvement in IC50 with combination of TAX and RA, with 1:100 being optimal. (C) Testing of RA, TAX, and RA-TAX loaded filomicelles on EC4 mouse liver cancer cells. TAX (black) has a much lower IC50 than RA (red) as it induces apoptosis instead of arresting proliferation. However, the combination of RA-TAX is more effective than either drug alone, with its IC50 less than half of that for TAX. Empty filomicelles (blue curve) were inert at the desired concentrations. (D) Nuclei treated with Paclitaxel (TAX) exhibit massive blebbed nuclei due to incomplete division (bottom). Untreated nuclei (top) in contrast are smooth and rounded. (E) Schematic depicting culture of resistant colonies arising after RA-TAX filomicelle treatment. (F) Plotting the proliferation rate against the degree of resistance gives an inverse relation, supporting the hypothesis that acquisition of drug-resistance occurs at the cost of proliferation.

Figure 6.

(A) RA and TAX treated cells were found to have higher levels of lamin-A than untreated ones, suggesting differentiation. Lamin-A levels were higher for drugs delivered via nanocarriers than for free drugs. (B,C) RA-TAX treated cells have lower levels of and Cyclin-D1 than TAX or RA alone treated cells. The durability of RA-TAX combination might be explained by its inhibitory role on protein synthesis (via Ki-67) rather than regulation of cell cycle (CyclinD1). (D) Images (40× magnification) of cells treated with RA, TAX, and RA-TAX combination, as well as untreated. All groups except RA-TAX stain positive for Ki-67. Lamin-A is higher in RA-TAX treated cells. (E) Delivery of drugs via filomicelles has been proven to be superior to free drugs, and this mode further reduces levels of these two proteins, with Ki-67 levels being halved compared to free drug treated. (F,G) histogram of Ki-67 and Cyclin D1 levels after treatment with various drugs loaded onto filomicelles. The fractions of cells null for these proteins progressively increases from no treatment to free drug to micellar drug delivery.

Figure 7.

(A) Quantifying RA effect on human derived induced pluripotent stem cells that were differentiated into mesenchymal stem cell lineage (iPS-MSCs). (B) Exposure to 10 nM TAX for 72 h blocks proliferation of both A549s and iPS-MSCs, as indicated by the decrease in cell number and proportion of Ki67 positive cells. In addition to the floating cells count, cell viability was also quantified by live/dead staining using Calcein-AM (live) and Ethidium Homodimer (dead). After the 10 nM TAX treatment, viability of the A549s is clearly more compromised (*p < 0.05 against DMSO).