Enhanced Uptake of a Thermally Responsive Polypeptide by Tumor Cells in Response to Its Hyperthermia-mediated Phase Transition¹

The limited therapeutic activity and insolubility of current anticancer drugs, toxicity and immunogenicity of the carrier, and inaccessibility and heterogeneity of the tumor provide a compelling rationale for the development of new drug delivery modalities for cancer therapy. Many different systems such as low molecular weight prodrugs, liposomes, and micro- and nanoparticles have been developed (1, 2, 3, 4, 5) in response to this need, with varying degrees of clinical success.

We are especially interested in combining macromolecular carriers with focused hyperthermia of solid tumors for a number of reasons. First, soluble polymeric carriers are intrinsically attractive for systemic drug delivery because polymer-drug conjugates preferentially accumulate in tumors due to the enhanced permeability and retention effect (6, 7, 8, 9, 10, 11) and also exhibit significantly lower systemic toxicity compared to free drug (12, 13, 14). In addition, studies have shown that water-soluble polymer carriers can overcome multidrug resistance (8, 15, 16, 17, 18). The most compelling evidence for the advantages of using polymer-drug conjugates over free chemotherapeutic agents for the treatment of cancer comes from extensive preclinical and clinical studies (Ref. 6 and the references within) on the use of N-(2-hydroxypropyl) methacrylamide copolymers as drug carriers. Second, hyperthermia preferentially increases the permeability of tumor vasculature compared to normal vasculature, which can further enhance the delivery of drugs to tumors (19, 20). To amplify the delivery of a polymeric drug in response to hyperthermia, we hypothesized that thermal targeting of polymeric drug carriers might offer additional synergistic advantages by appropriate choice of the polymeric carrier and hyperthermia treatment.

We have therefore investigated the feasibility of targeted delivery of systemically injected cancer therapeutics to heated tumors using thermally responsive biopolymer carriers consisting of a Val-Pro-Gly-Xaa-Gly (VPGXG) repeat unit, which we have named ELPs³ (21, 22, 23). We selected ELPs as polymeric drug carriers for these studies because they undergo an inverse temperature transition. Below a characteristic inverse transition temperature (T_t), ELPs are structurally disordered and highly solvated, but when the temperature is raised above their T_t, they undergo a sharp (2°C–3°C range) phase transition, leading to desolvation and aggregation of the biopolymer (21, 22). We hypothesized that the thermally triggered phase transition of ELPs might further enhance the accumulation of cancer therapeutics in heated tumors because of selective aggregation, in addition to accumulation of the drug-ELP conjugate in the tumor due to the enhanced permeability and retention effect and hyperthermia.

ELPs are also attractive for targeted drug delivery from a molecular design perspective because they are genetically encodable, which enables three important properties to be controlled to an extent that is impossible with synthetic polymers analogues. The synthesis of ELPs by recombinant DNA methods provides exquisite control over the ELP sequence and molecular weight. Control of these two parameters is critical to the design of carriers that undergo their inverse transition between 39°C and 41°C, a temperature range that is preferred for clinical applications of hyperthermia because it minimizes the incidence of edema and necrosis in healthy tissue surrounding a heated tumor (24). Control of macromolecular chain length and polydispersity is also important because it controls the residence time of the drug-polymer conjugate in systemic circulation (2). Genetically encoded synthesis also enables the type, number, and location(s) of reactive sites suitable for conjugation or chelation of drugs to be precisely specified along the polymer chain.

A previous in vivo study of ELP delivery to human tumors implanted in nude mice demonstrated that hyperthermia of the tumor resulted in a 2-fold increase in tumor localization of a thermally responsive ELP compared to localization without hyperthermia (25). Over half of the increased accumulation could be attributed to the thermally triggered aggregation of the ELP caused by the phase transition of the ELP in response to hyperthermia.

Although these results on tumor localization of ELPs are promising, we recognize that to demonstrate therapeutic efficacy, any targeting modality must successfully overcome three transport barriers to delivery: (a) targeting of the drug-carrier conjugate to the tumor microvasculature; (b) extravasation from the tumor vasculature into the tumor interstitium; and (c) transport of the drug to the appropriate molecular site of action within the target cells (26). Although this previous study addressed in part the question of whether the first two transport barriers could be overcome by this new targeting modality, the subcellular localization of ELPs has not been previously investigated. Because cellular uptake of the drug-polymer conjugate is a necessary prerequisite to its localization at the molecular site of action, we report here the cellular uptake of a thermally responsive ELP as a function of hyperthermia in three different tumor lines by flow cytometry and its subcellular distribution by confocal fluorescence microscopy.

The design and synthesis of ELPs have been described elsewhere.⁴ Briefly, genes encoding two different ELP sequences were synthesized: (a) one with guest residues Val, Gly, and Ala in a 5:3:2 ratio, respectively (termed ELP1); and (b) a control ELP in which this ratio was 1:7:8 (termed ELP2). These guest residue ratios were selected based on the previous results of Urry et al. (22), who characterized the effect of substitution of different amino acids at the fourth, “guest residue” position on the inverse transition of the ELP. ELP1 was designed to have a T_t of ∼40°C in aqueous solution to serve as a thermally responsive polymer under hyperthermia, whereas ELP2 was designed to have a substantially higher T_t (∼55°C) and serve as a thermally inactive control ELP.

Short gene segments (encoding 10 pentapeptides for ELP1 and 16 pentapeptides for ELP2) were constructed from chemically synthesized oligonucleotides (Integrated DNA Technologies, Inc., Coralville, IA) and initially cloned into pUC19 (New England Biolabs, Inc., Beverly, MA). The genes were oligomerized (resulting in genes encoding 150 ELP pentapeptides for ELP1 and genes encoding 160 pentapeptides for ELP2) and then cloned into a modified pET25b (Novagen, Inc., Madison, WI) expression vector. The expression vector contained translation initiation and termination codons and codons for short leading (Ser-Lys-Gly-Pro-Gly) and trailing (Trp-Pro) sequences. The ELPs were expressed in the Escherichia coli strain BLR(DE3) (Novagen, Inc.). The ELPs were purified from other E. coli proteins in the soluble fraction of the cell lysate by inverse transition cycling⁴ (25) and stored frozen at −80°C. The molecular weight of ELP1 was 59,200, and that of ELP2 was 61,100.

Fluorescence labels were conjugated to the primary amines of ELP1 and ELP1 using succinimidyl ester chemistry.⁴ Fluorescein-5-EX succinimidyl ester (Molecular Probes, Inc., Eugene, OR) was used to label the ELPs for flow cytometry experiments. For the cellular uptake and visualization of ELP distribution by confocal microscopy, ELPs were conjugated with Rhodamine Red-X succinimidyl ester 5-isomer (Molecular Probes, Inc.). The fluorophore:ELP ratio was ∼0.5 for both labels.

In separate experiments, a 15 μm solution of ELP1 or ELP2 in DPBS buffer was incubated with cathepsin B (1.4 units/ml) and cathepsin D [10 units/ml (pH 5.0)], as described previously (27, 28). After incubation for 1 h, the mixture was assayed by SDS-PAGE and visualized by copper staining (29). The detachment of the fluorescence label in the lysosomal compartment was similarly examined by incubation of ELP-rhodamine with lysosomal enzymes as described above. Rhodamine was visualized on the SDS-PAGE gel under UV transillumination.

The inverse transition of the ELPs and ELP-fluorophore conjugates was characterized by monitoring the absorbance at 350 nm as a function of temperature on a UV-visible spectrophotometer equipped with a multicell thermoelectric temperature controller (Cary 300 Bio; Varian, Inc., Melbourne, Australia). Reversibility of the inverse transition was examined by heating and then cooling an aqueous solution of ELP1 or ELP2 at a rate of 1°C/min. The inverse T_t was defined as the temperature at 5% maximum turbidity on heating an aqueous solution of an ELP.

DLS measurements were performed on a DynaPro LSR instrument (Protein Solutions, Charlottesville, VA). The intensity versus intensity time correlation functions were measured at a scattering angle of 90° as a function of temperature from 20°C to 65°C. At each temperature, the sample was allowed to equilibrate for ∼5 min before light scattering measurements were taken. Before use, the solutions were filtered through Whatman filters with a 0.02-μm pore size.

Human ovarian carcinoma (SKOV-3) and squamous cell carcinoma (FaDu) cell lines were grown as monolayers in 75-cm² tissue culture flasks containing RPMI 1640 (Roswell Park Memorial Institute) supplemented with 10% heat-inactivated fetal bovine serum, 2 mm l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (all from Life Technologies, Inc., Grand Island, NY). HeLa cells were grown in a culture medium composed of DMEM containing 10% FCS, as described previously (30). All three cell lines were cultured at 37°C in 5% CO₂ in air. The cultures were split 1:5 at confluence and passaged every 3–5 days.

Cells were removed from tissue culture flasks by brief treatment with 0.05% (v/v) trypsin-EDTA (Life Technologies, Inc.) and plated onto glass coverslips (22 mm; Corning Inc., Big Flats, NY) in a tissue culture dish (35 × 10 mm; Becton Dickinson Labware, Franklin Lakes, NJ) for confocal microscopy experiments or in 12.5-cm² tissue culture flasks (Becton Dickinson Labware) for flow cytometry experiments. At 1–2 days after plating, cells were incubated with either an ELP labeled with fluorescein or rhodamine, rhodamine-dextran (average molecular weight, 70,000; Sigma Chemical Co.), or fluorescent microspheres. The solution concentration of the ELPs and dextran was between 5 and 15 μm and is specified in the text or figure legends. For cell uptake of nano- and microparticles, fluorescence-labeled latex microspheres [Fluo-Spheres of 100 nm in diameter (catalogue number L5221) or 1 μm in diameter (catalogue number L5281; Molecular Probes Inc.)] were used as received. A 2% (v/v) suspension of the microspheres was sonicated for 5 min at room temperature and added to the cell culture medium to a final dilution of 0.05% (equivalent to 3.25 × 10⁹ microspheres/ml) of the original stock solution.

The cells on glass slides and in tissue culture flasks were incubated with gentle shaking at either the control or normothermic temperature (37°C) or heated to a temperature above the inverse transition temperature of the thermally responsive ELP1 carrier (typically 43°C to 45°C). The solution temperature was monitored by a thermocouple (Microprocessor thermometer type J-K-T thermocouple; Omega Engineering Inc., Stamford, CT). After incubation for 50 min, cells were gently rinsed with DPBS (Life Technologies, Inc.). The cells on glass slides were fixed for 10 min in 2% formaldehyde, mounted on glass slides (Propper Manufacturing Co., Inc., Long Island City, NY), and then visualized by confocal fluorescence microscopy. The cells in tissue culture flasks were removed from the flasks by brief treatment with 0.05% trypsin-EDTA (Life Technologies, Inc.). In some experiments, trypan blue, an extracellular fluorescence-quenching dye, was added at this step to differentiate between membrane-bound and internalized ELPs. Equal volumes of the trypsinized, resuspended cell suspension and a stock solution of trypan blue (0.5% in 0.85% NaCl) were allowed to mix for 5 min. Negative controls consisted of samples incubated in medium without added ELPs. The cell suspension was then centrifuged at room temperature for 5 min at 200 × g to remove cell debris and trypsin. The remaining cell pellet was resuspended in 2% formaldehyde for 10 min and centrifuged again. The cells were then washed briefly in DPBS and resuspended in 1 ml of DPBS, yielding a suspension suitable for analysis by flow cytometry.

The cellular uptake of fluorescence-labeled ELPs, dextran, or latex microspheres was measured by a fluorescence-activated cell scanner (FACS; Becton Dickinson, San Jose, CA) equipped with an argon-ion laser. The emission of fluorescein and rhodamine labels was measured using 530 and 585 bandpass filters, respectively. The analysis was performed using CellQuest software (Becton Dickinson). At least 10,000 events were acquired per sample. Forward and side light scatter gates were normally set to exclude dead cells, debris, or clumps of cells, unless otherwise stated. Cellular autofluorescence was measured using unlabeled cells.

The uptake of fluorescein-labeled ELPs was determined by calibrating the flow cytometer using a fluorescein standard kit (Flow Cytometry Standards Corp., Fishers, IN). Each fluorescein standard kit contains a set of beads with different levels of fluorescence intensity and a reference, blank population that is not labeled with a fluorophore. Each population in the set has a known number of fluorophores/bead. All microbeads are of the same size (approximately 7–9 μm in diameter), produce a single light scatter population, and appear within the same window of analysis. These quantitative fluorescence standards allow the calibration of the fluorescence intensity in flow cytometry in terms of the number of MESF.

Tumor cells that had been incubated previously with a fluorescence-labeled conjugate (ELP, dextran, or microspheres) and fixed with formaldehyde on glass microscope slides were imaged on a LSM-410 confocal microscope (Zeiss, Jena, Germany) equipped with a 488 nm argon and a 543 mm He/Ne laser and an Axiovert 135M (Zeiss) microscope with a ×100 oil immersion objective with 1.4 numerical aperture and an automated scanning stage (Zeiss). For the dual-stained preparations (e.g., cells coincubated with fluorescein-labeled microspheres and rhodamine-dextran) images for each fluorophore were independently acquired by two different photomultiplier tubes.

ELPs undergo a reversible phase transition in aqueous solution. They are hydrophilic and soluble in aqueous solution below their T_t, but they become hydrophobic and aggregate when the temperature is raised above their T_t. Therefore by exploiting the thermally triggered aggregation of ELPs, we hypothesized that ELP-drug conjugates can be targeted to solid tumors by selectively heating the tumor to a temperature above the T_t of the ELP.

In a previous study, we investigated the hyperthermia-mediated delivery of ELPs within solid tumors implanted in nude mice (25). We observed aggregates of a thermally responsive ELP by fluorescence videomicroscopy within the heated tumor microvasculature but not in normothermic tumors, which demonstrated that the phase transition of the thermally responsive ELP carrier could be triggered in vivo at a specified temperature. Importantly, we demonstrated that thermal targeting provides a ∼2-fold greater tumor localization compared with a thermally insensitive control polypeptide by in vivo videomicroscopy as well as by tissue distribution studies using radiolabeled ELPs. In vivo fluorescence videomicroscopy of implanted human ovarian tumors in nude mice also showed significant extravasation of the thermally responsive ELP into the interstitium in heated tumors. These studies clearly indicated that thermal targeting of ELPs by triggering the phase transition of the polypeptide carrier selectively in heated tumors enabled targeting of the drug-carrier conjugate to the tumor microvasculature as well as its extravasation into tumor tissue (25). Although these studies indicated that the thermally triggered phase transition of ELPs can thermally target ELP-drug conjugates to solid tumors, the success of ELP-mediated targeted drug delivery will require efficient cellular uptake and subcellular trafficking of the polymer-drug conjugate.

We therefore measured the uptake of fluorescein-labeled ELP as a function of solution temperature and ELP concentration using flow cytometry. To quantitatively compare the cellular uptake of ELP1-fluorescein in different cell lines as a function of temperature and concentration, we created a calibration between the cellular fluorescence intensity and the concentration of fluorophore using a set of calibrated fluorescence standards (see “Materials and Methods”). The conversion of fluorescence intensity to fluorophore concentration by this calibration procedure allowed quantitative comparison of ELP uptake as a function of ELP concentration and solution temperature across different cell lines.

For each cell line, the uptake of thermally responsive ELP1 was determined by flow cytometry for six different combinations of variables: three different concentrations of ELP1-fluorescein (5, 10, and 15 μm) at two temperature’s (T = 37°C < T_t and T = 43°C > T_t). We chose to examine the uptake of ELP1 in this concentration range because the T_t values of ELPs are an inverse logarithmic function of concentration (25). We have previously shown that ELP1-rhodamine conjugates display a transition temperature between 39°C and 41°C under physiological conditions for a solution concentration between 5 and 15 μm (25). Examining the cellular uptake of ELP1 in this range of solution concentration ensures that ELP1 does not undergo its phase transition in normothermic cells maintained at the physiological temperature of 37°C but undergoes its transition in cells heated to a temperature of 42°C or higher.

The uptake of ELP1-fluorescein at a solution concentration of 5 μm by human ovarian carcinoma cells (SKOV-3) was the same in heated cells (T > T_t) and in the control [cells maintained at the physiological temperature of 37°C (Fig. 1,A)]. Increasing the concentration of ELP1 to 10 or 15 μm resulted in a marked increase in cellular uptake in heated cells compared with the control cells maintained at 37°C [P < 0.05 (10 μm) and P < 0.01 (15 μm), heated versus normothermic SKOV-3 cells, unpaired t test]. Similarly, incubation of HeLa cells with 5 μm ELP1-fluorescein above the T_t had no effect on its uptake compared with incubation at 37°C (Fig. 1,B). However, when the concentration of ELP1-fluorescein was increased from 5 μm to 10 or 15 μm, the uptake of ELP1 was significantly greater in heated HeLa cells than in normothermic cells [P < 0.01 (10 μm) and P < 0.05 (15 μm), heated versus normothermic HeLa cells]. We also measured the uptake of ELP1-fluorescein in a squamous cell carcinoma (FaDu) as a function of concentration. As shown in Fig. 1 C, with increasing concentration of ELP1-fluorescein, there was a monotonic increase in the cellular uptake at both temperatures. Furthermore, at all concentrations between 5 and 15 μm, the uptake of ELP1 was significantly greater when the cells were heated to T > T_t, as compared with 37°C, with a 2-fold greater uptake with heat at 15 μm (P < 0.05 for 5 μm and P < 0.01 for 10 and 15 μm).

To confirm that the thermally responsive ELP1-fluorescein was internalized and not merely attached to the cell exterior, we used a trypan blue quenching assay to extinguish extracellular fluorescence (31). This assay is based on the observation that trypan blue quenches the fluorescence of fluorescein-conjugates attached to the cell membrane, whereas internalized fluorophores remain fluorescent. The fluorescence of ELP1-fluorescein decreased 20% in SKOV-3 and FaDu cell lines and decreased 40% in HeLa cells after trypan blue quenching, confirming that 80% of ELP1 associated with SKOV-3 and FaDu cells and 60% of ELP1 associated with HeLa cells was internalized (Fig. 1, D and E). This decrease in fluorescence was not dependent on the concentration of ELP1 or on the incubation temperature.

In summary, these results show that increasing the solution temperature above the T_t of ELP1 stimulated its uptake at a solution concentration of 10 and 15 μm in all cell lines relative to the control temperature of 37°C. These results clearly demonstrate that cellular uptake of a thermally sensitive ELP is enhanced by hyperthermia and that this effect is further modulated by the cell line and the ELP concentration.

We next dissected the role of heat in stimulating the uptake of the thermally responsive ELP1 by tumor cells. Because hyperthermia itself is likely to affect cellular uptake independent of the ELP phase transition, we sought to discriminate its effect on cellular uptake from that of the thermally triggered phase transition of the ELP. In control experiments, we therefore independently determined the uptake of a fluorescein-labeled thermally nonresponsive control polypeptide (ELP2) in all three cell lines above the T_t. ELP2 is a useful control for the effect of hyperthermia because it has a composition and molecular weight similar to those of ELP1, but it has a transition temperature significantly greater than the hyperthermia temperature (ELP2 T_t = 70°C; Ref. 25) and therefore does not undergo its phase transition when the cell culture medium is heated to 43°C.

As shown in Fig. 2,A, at a concentration of 5 μm, the uptake of ELP1-fluorescein by SKOV-3 cells heated to 42°C was statistically indistinguishable from that of the thermally insensitive control, ELP2-fluorescein, at the same temperature. At higher concentrations of 10 or 15 μm, however, the uptake of ELP1 was significantly higher than the uptake of the control, ELP2, under the influence of hyperthermia (P < 0.05, ELP1 versus ELP2). Similarly, in HeLa cells, there was no difference in uptake between the thermally active ELP1 and the control, ELP2, at an ELP concentration of 5 μm (Fig. 2,B). However, at 10 and 15 μm, cellular uptake of ELP1 was more than 80% and 30% greater than ELP2, respectively (P < 0.05). The FaDu cell line showed a significantly higher uptake of active ELP1 carrier than control, ELP2 carrier, at all concentrations with heat (P < 0.05; Fig. 2 C).

We interpret these results as follows: because ELP2 does not undergo its phase transition in heated cells, the absolute uptake of ELP2 in heated cells is controlled only by the nonspecific effect of hyperthermia, whereas the uptake of ELP1 in heated cells is controlled by both the effects of hyperthermia and its phase transition. Differences in the uptake of ELP1 compared with ELP2 in heated cells can therefore be attributed to the phase transition of the thermally active ELP1. In particular, the marked increase of the uptake of ELP1 in all three cell lines at 10 and 15 μm concentrations under the influence of hyperthermia can be attributed to the fact that under these conditions, the contribution of the ELP phase transition dominates the absolute uptake of ELP1 over the nonspecific effect of hyperthermia.

We distinguish between these two variables because the cellular effects of hyperthermia are likely to affect the uptake of all macromolecular carriers, even thermally nonresponsive polymers that do not alter their physicochemical properties in response to heat. In contrast, several important properties of thermally responsive polymers are altered on undergoing their phase transition, such as their degree of hydration (32) and hydrodynamic size,⁵ factors that are also likely to affect their interaction with cells and therefore affect their uptake. Next, we investigated the thermally triggered aggregation behavior of the ELP1 caused by its phase transition to further elucidate the role of the phase transition in stimulating its hyperthermia-mediated cellular uptake.

In response to the phase transition, soluble ELPs undergo a hydrophilic to hydrophobic phase transition, which results in the formation of ELP aggregates (22, 25, 33). We measured the change in the hydrodynamic dimensions of ELP1 during its phase transition by DLS (34). Fig. 3 shows the effect of the increase in temperature on the hydrodynamic radius of the thermally sensitive ELP1 at a concentration of 10 μm. Below its T_t, ELP1 was soluble, and its hydrodynamic radius (R_H) was ∼7 nm. An increase in the R_H to 100 nm was observed on increase in temperature above the critical temperature of polymer collapse, which was ∼40°C. A further increase in temperature resulted in a continued increase in the size of aggregates, resulting in the formation of aggregates with a steady state R_H of ∼1 μm. These results indicate that as the temperature is increased, the thermally active ELP1 undergoes a two-stage phase transition from soluble monomer to nanoparticles with a R_H of 100 nm and, finally, to large micrometer-sized aggregates. The hydrodynamic radii for all three species at an ELP concentration of 5 and 15 μm were identical to those shown in Fig. 3 for 10 μm, and the only difference in the temperature-dependent hydrodynamic properties of ELP1 between the three concentrations was the decrease in the T_t with dilution, as observed previously.⁴ This downward shift in T_t does not affect the occurrence of the phase transition because it is within the 37°C to 42°C range at all three concentrations.

Because the molecular weight and hydrodynamic size of drug carriers are important parameters in determining their cellular uptake by target cells (35), we hypothesized that the enhanced uptake of ELP1 by tumor cells in response to hyperthermia is related to the change in the hydrophobicity of ELP1 and its hydrodynamic size in response to temperature. We therefore investigated the dependence of cellular uptake on macromolecular size by confocal fluorescence microscopy in an effort to qualitatively elucidate the physical basis of the enhanced uptake of ELP1 observed in the three different cell lines under the influence of hyperthermia.

The top row in Fig. 4 shows confocal fluorescence images of ELP1-rhodamine in SKOV-3, HeLa, and FaDu cells after incubation of the cells above the T_t of ELP1. In all three cell lines, the cytoplasm was stained uniformly, and no fluorescent particles were observed below the T_t of ELP1 (Fig. 4, bottom row). In contrast, above the T_t, fluorescent particles were observed throughout the cytoplasm, suggesting that these particles are aggregates of ELP1 resulting from the ELP phase transition. Similar uniform cytoplasmic distribution in all three cell lines was observed for the thermally nonresponsive ELP2 with and without hyperthermia (data not shown).

We examined the susceptibility of ELPs to hydrolytic degradation by cathepsin B and cathepsin D, which are believed to play a major role in the lysosomal degradation of internalized proteins (28). In separate experiments, we incubated the thermally sensitive ELP1 and the control ELP2 with cathepsin B and cathepsin D as described previously (27, 28). No degradation products were observed by SDS-PAGE, suggesting that ELPs are not susceptible to hydrolysis. We also examined detachment of the fluorescence label in the lysosomal compartment as a possible cause for the uniform distribution of the ELP-rhodamine conjugate below its T_t by incubating ELP1-rhodamine with a mixture of both lysosomal enzymes. SDS-PAGE showed that ELP1-rhodamine and ELP2-rhodamine were present as a single distinct band with no measurable traces of free rhodamine, indicating that there is no detachment of the fluorescence label within the 1-h time frame of the ELP uptake experiments.

The uniform cytoplasmic staining below the T_t indicates that ELP monomers are taken up by tumor cells. In contrast, when cells are heated to a temperature above the T_t, in addition to the uptake of desolvated ELP monomer indicated by the background fluorescence in all three cell lines (Fig. 4, bottom panel), the presence of bright fluorescent particles can be attributed to the following possibilities: (a) ELP aggregates that are directly phagocytosed by cells from the culture medium; (b) desolvated ELP monomers that are concentrated within the cells and undergo their phase transition after uptake; or (c) preferential localization of desolvated, hydrophobic ELP monomers within subcellular compartments.

To qualitatively elucidate the relative contribution of each ELP species to the total uptake of ELP1 in the different cell lines and to investigate the origin of these bright particles of ELP1 in the cytoplasm of heated cells, we independently examined the uptake of model species that mimic the physicochemical properties of the three different populations of ELP1 that can coexist in the 37°C to 42°C temperature range.

We used dextran with a molecular weight of 70,000 as a model for the solvated ELP monomer below its T_t for two reasons: (a) its molecular weight and hydrodynamic radius closely match those of the ELP monomer; and (b) dextrans are highly hydrophilic polymers and are therefore physicochemically similar to the solvated, hydrophilic state of the ELP monomer below its T_t. Similarly, we modeled the endocytosis of 100-nm ELP nanoparticles and 1-μm aggregates by fluorescence-labeled latex microspheres of 100 nm and 1 μm in diameter.

Fig. 5 shows fluorescence images of SKOV-3, HeLa, and FaDu cells with (top row) and without hyperthermia (bottom row) that were incubated with a mixture of rhodamine-dextran and 100-nm-diameter fluorescent spheres. Rhodamine-dextran was distributed uniformly throughout the cell cytoplasm, and it also accumulated in the nucleolus of all cell lines. Interestingly, confocal microscopy images showed that the dextran was in a perinuclear arrangement in FaDu cells as opposed to the homogeneous distribution of dextran observed in SKOV-3 and HeLa cells. We also measured the uptake of rhodamine-dextran by flow cytometry (data not shown) in the 5–15 μm concentration range. The uptake of rhodamine dextran was dependent on the cell line, with the lowest uptake observed for SKOV-3 cells, and the highest uptake observed for FaDu cells. These results are qualitatively similar to the uptake of ELP1 by the different cell lines under the influence of hyperthermia. The cellular uptake of rhodamine-dextran also increased as a function of concentration in all three cell lines, a feature that was only observed for the uptake of ELP1 in FaDu cells. Simultaneously, with a filter set for fluorescein, we acquired fluorescence images of fluorescein-labeled 100-nm spheres (yellow). Whereas some of the fluorescent particles were only attached to the cell surface (bright yellow spots on the cells), a fraction of the 100-nm particles was also internalized (dim yellow spots inside the cells). However, none of the 1-μm microspheres were internalized in any of the cell lines (Fig. 6). Fluorescent particles were associated with the cells incubated above (Fig. 6, top row) and below the T_t, but they were attached only to the extracellular side of the cell membrane. Flow cytometry in combination with trypan blue quenching of extracellular fluorescence (results not shown) confirmed that SKOV-3, HeLa, and FaDu cell lines internalized fluorescent dextran with a molecular weight of 70,000 and 100-nm-diameter particles, but not 1.0-μm-diameter particles.

We believe that the enhanced uptake of ELP1 by heated cells compared with the uptake of the same polypeptide without heat or compared with the uptake of the thermally insensitive control ELP2 by heated cells is caused by increased uptake of hydrophobic ELP1 monomers and 100-nm particles, but not by direct uptake of 1-μm-diameter aggregates. Furthermore, because fluorescent particles are not observed in unheated cells and because large, 1-μm-diameter aggregates are not directly endocytosed by tumor cells, the fluorescent particles of ELP1 observed within heated cells are due to either the aggregation of ELP1 in the cytoplasm after uptake or preferential localization of hydrophobic ELP1 in subcellular compartments such as endosomes and/or lysosomes in heated cells. Our results do not allow us to distinguish between these possibilities.

This selective uptake based on size qualitatively observed by confocal microscopy and confirmed by flow cytometry indicates that ELP1 is internalized by a pinocytic rather than phagocytic mechanism of endocytosis (36). ELP1 becomes significantly more hydrophobic on undergoing its phase transition between 37°C and 42°C due to loss of bound water, as shown by microwave dielectric relaxation (37) studies and molecular dynamics simulations (32). We speculate that above the T_t, desolvated, hydrophobic ELP1 monomer and 100-nm nanoparticles show preferential adhesion to the cell membrane compared with hydrophilic, solvated ELP, which is the sole species present below the T_t. An adsorptive pinocytic mechanism (38) might show a higher uptake of the ELP compared with uptake solely by constitutive fluid-phase endocytosis (36) because of a higher local concentration of membrane-bound hydrophobic ELP monomers and nanoparticles of ELP1 compared with its solution concentration.

Finally, differences in the uptake of the thermally responsive ELP1 by the three different cell types may be caused by the different phospholipid and protein composition of the plasma membrane in different tumor cell lines that might exhibit differential adhesion of ELPs. This hypothesis is supported by our finding that in SKOV-3 and FaDu cells, only 20% of the ELP1-fluorescein associated with the cells was attached to the plasma membrane and was not internalized. For HeLa cells, in contrast, >40% of the total ELP1-fluorescein was not internalized, suggesting that differential endocytosis demonstrated in the above experiments may be associated with the differential adhesion of the hydrophobic, desolvated monomers and nanoparticles of ELP1 to the cell surface.

We have shown in this study that cellular uptake of a thermally responsive ELP in three different tumor lines increased significantly in response to hyperthermia. A significant fraction of the enhanced uptake of the thermally responsive ELP was attributable to its hyperthermia-mediated phase transition. We have also demonstrated that different cell lines exhibit different levels of uptake as a function of ELP concentration and heat, which may be associated with the differential adhesion of the ELP to the cell surface or with differences in the rate of pinocytosis with temperature. Confocal fluorescence microscopy revealed that tumor cells internalize the thermally sensitive ELP from their extracellular environment but that this uptake is limited to the ELP monomer and smaller aggregates in the 100-nm size range. These findings indicate that the enhanced uptake of the thermally responsive ELP by heated tumor cells is not caused by direct uptake of large, micrometer-sized ELP aggregates but by preferential uptake of desolvated, hydrophobic ELP monomers and smaller, 100-nm particles. These results also suggest that the design of the next generation of thermally sensitive ELP carriers should focus on sequences that form terminal aggregates that do not exceed a few hundred nanometers in diameter to maximize their cellular uptake.

Thermal targeting of ELPs yields an approximately 2-fold increase at the cellular level as compared with delivery under normothermic condition, as shown here, and a similar increase in localization at the tumor level as shown in a previous study (13). The likely additive effects of thermal targeting on tumor localization at the tissue and cellular level suggest that ELPs are a promising macromolecular carrier for thermally targeted delivery of cancer therapeutics. However, the significant difference in uptake of the thermally responsive ELP between different tumor cell lines as a function of hyperthermia also suggests that the development of thermally sensitive ELPs and other macromolecular carriers for delivery of cancer therapeutics should be optimized simultaneously not only at the whole tumor level via tissue distribution studies but also by optimization of its cellular uptake in the tumor cell line of interest.

We thank Dan Meyer for synthesis of the ELP gene and Dr. Kimberly Carlson for assistance with the DLS measurements.