The MDR human breast (MCF7TR) and ovarian (SKOV3TR) cancer cell lines have been previously established by selecting cells after exposure of the wild-type cultures (MCF7 and SKOV3) to continuous and increasing concentrations of PTX. The PTX IC50 for the MDR ovarian subculture (SKOV3TR) was over 100-fold higher at 1.08 μM than that of the drug-sensitive SKOV3 line, while similarly the PTX IC50 for the MCF7TR cells was tenfold higher at 0.98 μM than its drug-sensitive counterpart. To further verify that MDR phenotype was retained once the cells were xenografted in vivo and maintained throughout the entire study, the expression of the MDR marker proteins P-glycoprotein and GCS were examined. Figure reveals the expression of P-glycoprotein and GCS in cultures of SKOV3TR and MCF7TR cells, alongside their wild-type, drug-sensitive, parental lines prior to xenografting them into the mice to form tumors. During the duration of tumor growth, expression of these MDR markers was retained, as seen in Fig. by the evidence of P-glycoprotein and GCS staining in representative tumor tissues harvested from SKOV3TR- and MCF7TR-tumor-bearing mice that did not receive any treatment after a 4-week period.
Fig. 1 Drug-resistant SKOV3 ovarian and MCF7 breast tumor models. Expression of the MDR marker proteins P-glycoprotein and glucosylceramide synthase in SKOV3TR and MCFTR cells as compared with their wild-type cell lines a in culture observed by Western blot (more ...)
Prior in vitro
work fully examined the efficacy and mechanism of action of CER as a cotherapy to revert the MDR phenotype in these cultures (8
). Cell kill efficacy of both MDR cell types increased significantly when CER was administered alongside PTX, shown in previous work to result from reduction of the apoptotic threshold in MDR cancer cells (8
). It was found, however, that an interesting and important relationship existed between dosing of PTX and CER, in that cell kill efficacy increased significantly in both MDR lines when CER was administered with a delay of several hours following PTX administration; however, the converse did not hold true. For example, in MCF7TR
cells, cell survival decreased from 60% when the drugs were coadministered to 47.1% when CER was administered 6 h following CER (p
0.05). Interestingly, this increase was not observed when PTX was administered with a 6-h delay following CER (61.1% cell survival) in this cell line (Table ). Similar results were seen in the SKOV3TR
line, where dosing of CER 6 h after dosing of PTX decreased cell survival significantly (6.0% vs
. 8.7% when the drugs were coadministered, p
0.05), while temporally delivering the drug in the opposite sequence did nothing to improve efficacy (Table ). Interestingly, this same temporal dosing relationship did not improve cell kill efficacy in drug-sensitive MCF7 and SKOV3 cells. In fact, sequential delivery of CER followed later by PTX reduced the efficacy of this combination therapy in the MCF7 line. While it has repeatedly been shown that a PTX + CER combination therapy also enhances chemosensitivity of drug-sensitive cells, it is hypothesized that this result is in part an additive effect of the individual toxicities of two drugs, while we have proposed a synergistic mechanism of action for the two drugs in MDR cells (14
). This differential effect could lead to a dependence on kinetic dosing in MDR cells, while independent on kinetic dosing in the drug-sensitive population.
The Effect of Temporal Spacing Between PTX and CER on Cell Viability in MDR and Drug-Sensitive Cells
To incorporate this dose–kinetic relationship into the formulation, a nanoparticle system was designed that could simultaneously carry both the PTX and CER therapeutics but release each in a controlled manner within tumor cells upon uptake. In order to develop these multifunctional nanoparticles, the pH-responsive polymer PbAE was blended at 30% together with a slow-degrading polymer, PLGA, incorporated at 70%, with the intention that the polymer phases would remain immiscible, causing PbAE to form pH-responsive “pockets” or regions within the PLGA matrix (Fig. ), a strategy taken from previous work by Little and Langer et al.
). The 70% PLGA/30% PbAE ratio was experimentally derived by blending PLGA and PbAE together in various ratios ranging from 10:90% PLGA/PbAE to 90:10% PLGA/PbAE in increments of 10%. Based on particle size and drug loading efficiency, the 70% PLGA/30% PbAE blend was chosen for development of this nanoparticle platform. In the formulation described herein, PTX was loaded into the pH-sensitive regions, while CER was loaded into the PLGA matrix, so that PTX would be rapidly released upon internalization into the tumor environment caused by the drop in pH to 6.5, and CER release would slowly follow from the PLGA matrix. Scanning electron microscopy images reveal the production of spherical particles (Fig. ), while dynamic light scattering measured particles at an average size of 208
6 nm. Zeta potential for these particles averaged around −26.9
5.4 mV. Chemical surface analysis revealed that, as intended, the majority of PbAE was internalized into the nanoparticle matrix, since the atomic signature of the surface of the 70% PLGA/30% PbAE nanoparticles (at 61.5
0.3% carbon, 0.8%
0.3% nitrogen, and 37.8
0.6% oxygen) resembles that of pure PLGA (58.5
0.1% carbon, 0.0
0.0% nitrogen, and 43.6
6.2% oxygen), while an even blend of the two polymers would cause a surface signature to contain even traces of both polymers. PbAE is particularly characterized by the presence of nitrogen (5.7
0.5%) in the chemical makeup, as well as a higher percentage of carbon (73.6
0.7%) and a lower percentage of oxygen (16.6
0.1%) compared with PLGA. Since few traces of this PbAE chemical signature are detected on the surface of the nanoparticles, we concluded that PbAE was internalized into the PLGA particles as designed. The drug release profile, however, was the most conclusive evidence that the nanoparticles had been developed as hypothesized (Fig. ). Data revealed that PTX was released more rapidly than CER and exhibited a strong pH-responsive effect, whereby the remainder of the payload was immediately released upon decrease of pH to 6.5. On the other hand, only 60% of encapsulated CER was released by the time PTX egress had expired. Moreover, CER release exhibited only a minimal pH-responsive effect, suggesting that indeed the majority of CER is associated with the PLGA matrix, whereas PTX preferentially associates with the pH-responsive PbAE matrix.
Fig. 2 PbAE/PLGA polymer-blend nanoparticles for temporal-controlled delivery. a Illustration of polymer-blend nanoparticle design, whereby the pH-responsive poly(beta-amino ester) (PbAE) internalizes into a matrix of slow-releasing poly(d,l-lactice-co-glycolide) (more ...)
SKOV3 tumor mass was established by subcutaneous administration of cells in female nude mice. On the other hand, similar to clinical forms of human breast cancer, the MCF7 cancer cell line is estrogen dependent for proliferation (17
). Since intact female mice do not produce high enough estrogen levels to support MCF7 tumor development, mice must be supplied with exogenous estrogen through the insertion of continuous-release estrogen implants that maintain steady-state plasma estrogen level for at least 6 weeks. To prime the mice with sufficient estrogen to support tumor growth, silastic estradiol implants were prepared and inserted subcutaneously on the right dorsal side of intact female nu/nu mice, 48 h prior to tumor inoculation. The implants were designed to release estradiol into the bloodstream to maintain a steady-state level of 50 pg/ml for up to 6–8 weeks. While the estradiol implants indeed maintained a steady-state level at around 50 pg/ml at the start of treatment (2–3 weeks following implantation), this level had attenuated over the duration of treatment to settle at around 40 pg/ml by the end of the treatment period (data not shown).
To monitor the efficacy of this sequential PTX + CER combination treatment in blend nanoparticle formulations, tumor-bearing mice were randomly assigned to treatment with either control (untreated), PTX (free drug) at 20 mg/kg, and PTX + CER (free drug) at 20 mg/kg and 80 mg/kg, respectively, PTX in polymer-blend nanoparticles at 20 mg/kg, and PTX + CER in polymer-blend nanoparticles. Once the tumors had reached a palpable size of at least 100 mm3, the mice were given a single-dose intravenous injection of their assigned treatment, after which tumor volume was routinely measured with a metric Vernier caliper for 3 weeks. Figure shows the therapeutic response as percent change in tumor volume over time for SKOV3TR tumors (Fig. ) and MCF7TR tumors (Fig. ). In both tumor models, the experimental PTX + CER nanoparticle therapy was most efficacious. Although the PTX + CER treatment administered as free drug showed a delay in tumor growth early on by day 4 in the SKOV3TR mice (Fig. ; 30% tumor volume change from the PTX treated groups) ,this effect was quickly lost thereafter and was entirely absent in the MCF7TR tumors, which were not significantly responsive to PTX + CER as free drug (Fig. ). Similarly, the PTX + CER nanoparticle treatment quickly regressed tumor growth by day 4 to produce a 40% lower tumor volume change compared with the PTX-treated groups. Only in this treatment group was tumor growth delay significantly retained. The therapeutic results were quite similar for the MCF7TR tumors (Fig. ). Within this treatment set, the PTX + CER nanoparticle therapy shows the greatest trend towards tumor regression with a 36% drop in tumor volume from initial, although this result is only significantly different from the remaining drug treatment groups at day 21 and not as early on as after administration as seen in the SKOV3TR tumors. Unlike the SKOV3TR tumors, the PTX + CER (free drug) treatment had no significant effect on tumor growth reduction from control or PTX alone. However, around the second week after treatment administration, the PTX + CER nanoparticle therapy did significantly decrease tumor growth when compared to the PTX + CER free drug therapy, but not when compared to the PTX alone (either as free drug or nanoparticles).
Final tumor weight, measured 28 days following treatment initiation, also revealed that the PTX + CER nanoparticle therapy was the most efficacious treatment group for both tumor types (Fig. ). At an average of 31.2
1.1 mg, the final tumor weight in the SKOV3TR
mice after treatment with PTX/CER nanoparticles is significantly less than the final tumor weights of mice in not only the control group but also in groups that received any of the other drug treatments (PTX, PTX + CER, PTX nanoparticles; p
0.05, Fig. ). Similarly, in the MCF7TR
breast cancer model, again the PTX + CER nanoparticle treatment resulted in the smallest final tumor weight, at 21
3 mg, which is significantly lower than the final tumor weights of the other four treatment groups (Fig. ). Since the mice in all the treatment groups started out with, on average, equal initial tumor volumes (~100 mm3
), it is expected that final tumor weight would correlate well to treatment efficacy. When injected intravenously in suspension, blank nanoparticles did not have any antitumor effect.
While the tumor efficacy data on both MCF7TR and SKOV3R MDR models appears to support the hypothesis that the PTX + CER combination therapy delivered within the 70% PLGA/30% PbAE nanoparticles significantly enhances the ability to suppress the growth of MDR tumors, the question remains whether this therapy indeed accomplishes this feat by lowering the apoptotic threshold in MDR cancer cells, as determined in prior in vitro studies. To determine apoptotic activity in response to the various treatment groups, tumors harvested on day 28 after treatment were stained for apoptotic activity by TUNEL staining, which labels nick-end DNA fragments, one of the hallmarks of apoptotic activity, resulting in colorimetric output where apoptotic activity is present. Figure shows stained tumor sections from SKOV3TR tumors and from MCF7TR tumors. While the free drug PTX and free drug PTX + CER treatments showed minimal TUNEL staining in their sections apart from faint indications, the PTX + CER nanoparticle therapy caused the most intense TUNEL-positive staining on all of its tumor sections in both the SKOV3TR and the MCF7TR tumors. This result appears to suggest that the combination drug therapy in the polymer-blend nanoparticles is able to restore apoptotic activity in the MDR tumor cells to overcome MDR, since apoptotic activity is most overwhelming in the PTX/CER nanoparticle-treated tumor sections, supporting the conclusion of an enhanced therapeutic efficacy associated with this treatment.
Fig. 4 Enhancement in tumor apoptotic response by TUNEL staining. Tumor TUNEL staining to indicate enhancement in apoptotic activity in SKOV3TR and MCF7TR tumor sections, harvested at day 28 following a single-dose intravenous treatment with 20 mg/kg (more ...)
A key measure in the development of any new therapeutic is a preliminary evaluation of safety, especially with polymeric nanoparticle-based delivery. To evaluate safety and thereby any potential toxicity of the PTX + CER polymer-blend nanoparticle therapy, we examined body weight changes, white blood cell count, and serum liver enzyme activity over time following treatment initiation. Evidence for a lack of toxicity with this novel MDR therapy, changes in body weight (Table ), and white blood cell count (Table ) were also monitored for the duration of the treatment period, since significant increases in white blood cell counts and decreases in body weight are also indicative of toxicity. The lack of toxicity was verified when neither of the treatment groups causes a significant drop in body weight up to 4 weeks following treatment initiation in either MCF7TR and SKOV3TR tumor models (Table ). Even though it is observed that the Cremophore EL®-based soluble drug formulation causes a slight decrease in body weight around weeks 3 and 4 in MCF7TR-tumor-bearing mice treated with PTX + CER or PTX administered as soluble drug, respectively, this effect can be attributed to the Cremophore EL® (a known irritant) and has no bearing on the safety of the experimental PTX/CER polymer-blend nanoparticle therapy. Table demonstrates that there was lack of significant changes in white blood cell count following any of the treatment groups over time. No significant rises in white blood cell counts are observed in the SKOV3TR-tumor-bearing mice following treatment with either any the experimental PTX + CER nanoparticles or any control groups. The same result was seen in the MCF7TR tumor model where neither treatment group exhibits a trend towards elevation of white blood cell count. However, in this MCF7TR model, the PTX nanoparticle treatment shows a slight elevation in white blood cell count, corresponding to the elevation of serum LDH seen in these mice (Table ). However, this result is neither significantly elevated over basal nor significantly different from the untreated control.
Percent Body Weight Change Following Treatment with the Combination Therapy
WBC Counts Following Treatment with the Combination Therapy
Liver Enzyme Levels Following Treatment with the Combination Therapy
A standard method to evaluate acute liver toxicity resulting from systemic administration is to monitor increases in serum enzyme activity of ALT and LDH, as these indicate any acute toxicity to the liver. Table shows the changes in serum ALT and LDH activity for mice bearing SKOV3TR and MCF7TR tumors. While the results suggest that there is no overall toxicity of the nanoparticles as measured by serum enzyme levels, isolated incidents exist whereby activity increases following treatment. For example, although an increase is seen in serum ALT and LDH activity at day 28 after administration of the PTX + CER soluble drug treatment in SKOV3TR-tumor-bearing mice, this increase is not only completely absent in the MCF7TR-tumor-bearing mice but is also, in neither case, a significant change from basal level (day 1). Similarly, while the PTX nanoparticle treatment appears to double serum LDH activity by day 28 in the MCF7TR-tumor-bearing mice, this difference is again not significant from day 1.