Brachytherapy is a common clinical technique involving implantation of sealed radioactive “seeds” within a tumor to selectively irradiate the tumor mass while minimizing systemic toxicity. To mitigate the disadvantages associated with complex surgical implantation and subsequent device removal procedures, we have developed an alternative approach using a genetically encoded peptide polymer solution composed of a thermally responsive elastin-like polypeptide (ELP) radiolabeled with 131I that self-assembles into radionuclide seeds upon intratumoral injection. The formation of these nontoxic and biodegradable polymer seeds led to prolonged intratumoral retention (∼85% ID/tumor 7 days postinjection) of the radionuclide, elicited a tumor growth delay in 100% of the tumors in two human xenografts (FaDu and PC-3), and cured more than 67% of tumor-bearing animals after a single administration of labeled ELP. These results suggest that in situ self-assembly of biodegradable and injectable radionuclide-containing polypeptide seeds could be a promising therapeutic alternative to conventional brachytherapy. Cancer Res; 72(22); 5956–65. ©2012 AACR.

Brachytherapy is a clinical technique that is typically used in the treatment of prostate cancer and other malignancies to selectively expose a tumor to large doses of beta and gamma radiation via locally implanted “seeds” (1). The strategic placement of these seeds throughout the tumor mass can provide prolonged irradiation to the tumor while limiting the radiation burden on healthy tissues. Although the success of current approaches to prostate brachytherapy is exemplified by excellent disease-free survival (2–4), its implementation is plagued by a number of significant limitations, including the need for general anesthesia, complicated placement procedures, seed migration to other organs, and the need for posttreatment excision for seed removal (5). We speculated that many of these limitations might be overcome by engineering a biodegradable material that (i) can form injectable depots with a life span in vivo that is compatible with the half-life of the radionuclide used for treatment and (ii) degrades into nontoxic products that are resorbed by the body after the radioactivity has decayed to a level that no longer poses a threat of systemic toxicity, thereby obviating the need for surgical implantation and reexcision. Although various polymers have been designed and evaluated with these requirements in mind, issues such as toxicity, biologic incompatibility, a lack of injectability and the less ability to scale-up manufacturing continue to hinder their clinical implementation (6–9).

Elastin-like polypeptides (ELP) are a unique class of genetically engineered peptide polymers that have several attractive properties for brachytherapy. First, ELPs undergo phase separation in response to small temperature changes. ELPs remain highly soluble in aqueous solution below a characteristic transition temperature (Tt), but within seconds, desolvate into a hydrophobic coacervate above the Tt. This temperature is tunable by adjusting the amino acid composition, molecular weight (MW), and concentration of the ELP (10–12). The ability to tune the behavior at the molecular level allows for the facile design of ELPs that can serve as an injectable depot for radioactivity or other drugs. Second, ELPs are biodegradable, biocompatible, and their design at the genetic level enables facile addition of functional groups for radiolabeling (13–15) and provides exquisite control over the composition and MW of the peptide. This versatility facilitates the design of ELPs that are soluble at room temperature but rapidly collapse into a viscous coacervate upon warming to body temperature. This feature is attractive for the formation of an injectable depot of covalently conjugated radionuclide for brachytherapy, as it avoids the need for surgical placement of the radioactive depot. In a previous study, the ELP phase transition temperature was adjusted to drive ELP coacervation immediately following tumor infusion. The formation of a coacervate phase within the tumor increased the intratumoral retention of the radiotherapeutic, thereby enhancing the antitumor efficacy compared with a thermally insensitive control ELP. However, this [131I]ELP depot was not able to induce complete tumor regression (16).

Herein, we engineered an easily injectable radiolabeled ELP that thermally self-assembles into highly stable, therapeutic radionuclide seed-like depots in situ upon intratumoral (i.t.) administration and optimized their in vivo performance. To optimize the therapeutic efficacy of this new modality, we investigated the effect of a range of parameters to modulate the physical properties of the depot, including the ELP MW, concentration, composition, and the tyrosine-rich peptide appended to the carboxy-terminus for radiolabeling. By designing a diverse set of injectable ELPs, we show that the temporal stability of the radioactive depot in vivo can be tuned by controlling the phase transition temperature of the radionuclide carrier and present evidence of the therapeutic efficacy of this engineered thermally responsive biomaterial. The optimized seed-like depot displays prolonged intratumoral retention and exhibits potent antitumor efficacy, and eventually degrades into nontoxic peptides.

ELP design and synthesis

A thermally responsive ELP composed of tandem repeats of the pentapeptide motif VPGVG was designed to transition below 30°C to enable immediate coacervation upon intratumoral infusion within the concentration range of 62.5 to 1,000 μmol/L. To evaluate the effect of MW on tumor retention, synthetic genes encoding 3 ELPs spanning a range of MWs were synthesized using PRe-RDL (17). These 3 ELPs, consisting of 60 pentapeptides (25 kDa), 120 pentapeptides (50 kDa), and 240 pentapeptides (100 kDa), are referred to as ELP60, ELP120, and ELP240, respectively. ELP60 and ELP240 included a single tyrosine residue at the C-terminus, and are referred to as ELP60-Tyr1, and ELP240-Tyr1. ELP120 was modified to include 1, 4, or 7 tyrosine residues at the C-terminus, and these are referred to as ELP120-Tyr1, ELP120-Tyr4, and ELP120-Tyr7, respectively. Synthetic genes encoding these ELPs were inserted into pET-24a (+) expression vectors and transformed into BL21 Escherichia coli chemically competent cells (Edge BioSystems). Transformed cells were used to inoculate one liter of TB-Dry media (Mo Bio) in a 4-liter flask, which was supplemented with 45 μg/mL kanamycin and incubated for 24 hours at 37°C and 200 rpm. ELP was purified using inverse transition cycling (ITC; ref. 18), a nonchromatographic method that exploits the ELP thermal transition to sequentially remove soluble and insoluble impurities through centrifugation. ITC also enables the recovery of ELP at high yields with endotoxin levels that are below the FDA required limit of 5 EU/dose (1 dose = 1 mg). The ELP thermal properties were characterized by monitoring the optical density of the ELP solution at 350 nm as a function of temperature (1°C/min) on a temperature-controlled UV-Vis spectrophotometer (Cary 300 Bio; Varian instruments).

Radioiodination of ELP

ELPs with carboxy-terminal tyrosine residues were labeled with either 125I or 131I (PerkinElmer) using the IODO-Gen kit (GE Healthcare). Briefly, 100 μL of ELP at a specified concentration in PBS was applied to an IODO-Gen precoated tube containing 2 mCi Na 125I (for tumor retention and biodistribution studies) or 20 mCi Na 131I (for radiotherapy studies), and the reaction product was purified by size-exclusion chromatography with a PD-10 column (GE Healthcare). The radioactivity was quantified by counting an aliquot on a γ-counter (LKB-Wallac). The concentration of iodinated ELP was measured by UV-Vis spectrophotometry (Thermo Scientific) at a wavelength of 280 nm. The final radioactivity and molar concentration of ELP was adjusted by mixing labeled and unlabeled ELPs to achieve the desired injection concentration and dose. The radioactive dose of [125I]ELPs for tumor retention studies was 10 μCi 125I/40 μL of ELP with a specific radioactivity of 6 to 12 μCi/mg, whereas for biodistribution studies the dose was 3 μCi 125I/40 μL of ELP at 1,000 μmol/L with a specific radioactivity of 1 to 2 μCi/mg. To assess therapeutic efficacy, we used [131I]ELPs at 1,000 to 2,000 μCi 131I/40 μL of 1,000 μmol/L ELP with a specific radioactivity of 7,500 to 15,000 μCi/mg.

Animal implantation of tumor subcutaneous xenografts

Female nude mice (Balb/c, nu/nu) with an average body weight of 20 g were provided by the Duke Comprehensive Cancer Facility Isolated Facility for all in vivo experiments, including ELP tumor retention, biodistribution, and [131I]ELP radiotherapy. The mice were housed in isolated caging with sterile rodent food, acidified water ad libitum, and a 12-hour light/dark cycle. The FaDu head and neck squamous cell carcinoma cell line was obtained from the American Type Culture Collection and maintained by Duke University Cell Culture Facility. PC-3M-luciferase gene (luc2), which is a derivative of the PC-3 cell line, is a luciferase-expressing metastatic prostate cancer cell line that was stably transfected with firefly luc2 (Caliper Life Science). Both cell lines were authenticated by STR-DNA technology (RADIL) by Duke University Cell Culture Facility, and Caliper Life Science, respectively, when we bought them and started cell culture for this study in our laboratory. The 2 tumor cell lines were cultured as monolayers in tissue culture flasks. The culture medium contained minimal essential medium (MEM) supplemented with 10% heat-inactivated FBS and a 1% antibiotic/antimycotic solution (Invitrogen). The cultures were maintained at 37°C under 5% CO2. Leg tumor xenografts were established by subcutaneous (s.c.) inoculation of 1 × 106 cells in 30 μL of MEM medium in the right lower leg of each mouse. Tumors were allowed to grow for 7 to 9 (FaDu) or 11 to 13 (PC-3) days before treatment until they reached a size of 150 ± 20 mm3. Mice were monitored for general well being, weight, and tumor volume. All animal experiments were conducted in accordance with the Duke University Institutional Animal Care and Use Committee regulations.

Tumor retention of ELP conjugates

To investigate the effect of the injected concentration, MW, and tyrosine content on the tumor retention of ELP, 3 retention experiments were conducted, varying: (i) the injection concentration of ELP120-Tyr1 (62.5, 125, 250, 500, and 1,000 μmol/L); (ii) MW of the ELPs (ELP60-Tyr1, 25 kDa; ELP120-Tyr1, 50 kDa; and ELP240-Tyr1, 100 kDa); and (iii) the tyrosine content at the C-terminus of ELP120 at an injection concentration of 250 μmol/L (ELP120-Tyr1, ELP120-Tyr4, and ELP120-Tyr7). Each ELP was infused into the tumor using a syringe pump (Harvard Apparatus) at a rate of 120 μL/min in a volume of 40 μL into a 150 mm3 tumor. The radioactivity retained in the mice was monitored with a dose calibrator (Biodex Medical Systems) at 0, 24, 48, 72, 96, and 168 hours.

Rheological characterization

The ELP viscosity in PBS was measured on an AR G2 rheometer (TA Instruments) with a concentric cylinder. An ELP solution in PBS was loaded onto a preheated cylinder (35°C), the samples were compressed to a height of 55 μm and allowed to equilibrate for 2 minutes at 35°C. Steady shear measurements were conducted over a range of shear rate between approximately 10−3 and approximately 103 per second.

Dynamic light scattering

Dynamic light scattering (DLS) was conducted on ELP120-Tyr1, ELP120-Tyr4, and ELP120-Tyr7 using a thermally controlled Wyatt Plate Reader (Wyatt Technology) to determine the hydrodynamic radius (Rh). The samples were diluted to 50 μmol/L in PBS, filtered through a 0.1 μm Anotop syringe filter (Whatman), and 35 μL of each solution was placed into a well of a 384-well plate (Corning). Small drops of mineral oil were added to the top of each well to prevent evaporation. Each sample was analyzed with eighteen 10 second acquisitions at 15°C, which was below the transition temperature for these ELP. The resulting data were fit using a Rayleigh sphere model with a regularization algorithm. Populations comprising less than 3% of the total mass were excluded from analysis.

Tumor retention and therapeutic efficacy of [131I]ELPs

The effect of increased tyrosine content on the in vivo tumor retention and antitumor efficacy of [131I]ELP was examined in a s.c. FaDu tumor model by i.t. administration of the following ELP solutions: [131I]ELP120-Tyr1, [131I]ELP120-Tyr4, and [131I]ELP120-Tyr7 at an ELP concentration of 1,000 μmol/L and a total radioactivity dose of 2,000 μCi into a 150 mm3 tumor. The in vivo retention and antitumor efficacy of [131I]ELP120-Tyr7 was also examined in subcutaneous human prostate PC-3M-luc2 xenografts in nude mice. Both tumors were compared with an unlabeled ELP control, also administered at 1,000 μmol/L. To prevent thyroid accumulation of free radioiodine, 1% potassium iodide was added to the drinking water of all of the animals one week before treatment and continued till the end of the radiotherapy experiment. The mice were monitored daily for tumor radioactivity, tumor volume, body weight, and survival during the first week, every other day during the first month, and twice a week thereafter. The tumor radioactivity profile was monitored with dose calibrator after i.t. injection of [131I]ELP until the level fell below 5% of the administered level in each mouse. The tumor volume was determined by the equation: volume = (width)2 × length × π/6. The mean tumor growth over time per group was calculated and expressed as the relative tumor volume compared with day 0 (i.e., fold change). Median values were used instead of the mean because some tumors regressed completely. Animals were humanely killed once any of the following end points were reached in the course of the radiotherapy: the tumor size reached 5 times the initial tumor volume, the tumor volume reached 1 cm3, the body weight dropped below 85% of the body weight, or 60 days posttreatment (the end of the experiment).

Safety evaluation of ELPs

The safety evaluation of the ELP radionuclide depot was examined through 2 experiments: (i) the radioiodine toxicity during radiotherapy was evaluated by monitoring clinical signs of health and changes in body weight and (ii) the organ and tissue toxicity was examined by measuring the biodistribution of [125I]ELP120-Tyr7 after i.t. administration in nude mice bearing FaDu tumors. Six mice were sacrificed at predetermined time points, their organs (blood, thyroid, lungs, heart, liver, spleen, kidneys, and tumor) were collected, and the radioactivity levels were measured with a γ-counter. The accumulated radioactivity was presented as injected dose per gram (%ID/g).

Histologic studies and tumor autoradiography

On days 0, 1, 2, 3, and 60 after i.t. infusion of [131I]ELP, the animals were killed, and the tumor tissue was collected and fixed in 10% formalin. The formalin-fixed FaDu and PC-3 tumor tissue was then embedded in paraffin, sectioned (5 μm), and stained with hematoxylin eosin. Tumor necrosis in both treatment and control groups was analyzed by light microscopy. Unstained tumor sections from the same set of samples were exposed to a storage phosphor screen (PerkinElmer) for 4 to 5 days, which was then scanned with a Cyclone storage Phosphor (PerkinElmer)

Statistical methods

Tumor retention data of [125I]ELP and tumor growth data for [131I]ELP were analyzed by 1-factor ANOVA analysis stratified by treatment group, which was followed by the Bonferroni t test. P < 0.05 was considered statistically significant in both cases. Statistical differences between survival rates of the animals were determined by Kaplan–Meier analysis. The cumulative radioactivity and half-life of tumor radioactivity were analyzed with PK Solution 2.0 (Summit Research Services).

Modulation of the phase transition behavior enables prolonged intratumoral retention

To promote intratumoral retention, each ELP was designed to form highly localized insoluble depots—analogous to seeds—at the temperature of subcutaneous tumors in anesthetized mice (31.7°C). The Tt of each ELP satisfies this criterion under all examined conditions, including changes in concentration, MW, and tyrosine content (Supplementary Table S1). The injected ELP is expected to remain assembled in a localized seed until its degradation and alternatively clearance by diffusion.

To determine the relationship between tumor retention and the physical properties of the ELP, concentration, MW, and tyrosine content were systematically varied. First, the effect of concentration was examined on the in vitro Tt and in vivo tumor retention of ELP120-Tyr1 over a series of 2-fold dilutions (1,000–62.5 μmol/L). Although the Tt decreased 3°C over this 16-fold increase in concentration, the in vivo tumor retention 7 days following intratumoral infusion exhibited a 5-fold increase from 16% at 62.5 μmol/L to 85% at 1,000 μmol/L (P < 0.01; Supplemental Table S1). These results indicate that higher concentrations could provide a significant increase in antitumor efficacy by increasing the tumor retention time of radioiodine.

Second, the effect of the ELP MW on the in vitro Tt and in vivo tumor retention of the ELP seeds was examined using ELP60-Tyr1 (25 kDa), ELP120-Tyr1 (50 kDa), and ELP240-Tyr1 (100 kDa). The ELP Tt exhibited a 4°C decrease as the MW increased from 25 to 50 kDa; this effect also translated to higher tumor retention, which showed a 14-fold increase 7 days following infusion (P < 0.01). However, further increasing ELP MW to 100 kDa (ELP240-Tyr1) only reduced the Tt by an additional 0.5°C and failed to generate a significant increase in tumor retention from ELP120-Tyr1 to ELP240-Tyr1 (Supplemental Table S1). These results also suggest that the ELP Tt plays an important role in tumor retention. Thus, ELP120-Tyr1 (50 kDa) was selected for further characterization because it provided prolonged tumor retention while reducing the amount of ELP that must be administered.

Finally, we examined the effect of the number of tyrosine residues in the ELP on its tumor retention. It is generally understood that the ELP phase transition depends on the hydrophobicity of the sequence; as the hydrophobicity of the ELP increases, the Tt decreases (19). However, instead of altering the ELP sequence, a hydrophobic tyrosine-rich peptide was appended to the C-terminus to provide reactive sites for radioiodination as well as to decrease the Tt. As expected, the Tt of the ELPs decreased as a function of tyrosine content: ELP120-Tyr1 (24.97°C), ELP120-Tyr4 (23.22°C), and ELP120-Tyr7 (20.57°C) all followed this trend. This reduction in the Tt caused a significant increase in the tumor retention of the seed, with each addition of 3 tyrosine residues inducing a 1.5- to 2-fold increase in tumor retention and a 2.5°C reduction in the Tt (P < 0.01; Supplemental Table S1). Correlation analysis of the tumor retention experiments showed that each of the 3 factors plays a critical role in the Tt: ELP concentration (r2 = 0.983), ELP MW (r2 = 0.998), and tyrosine content (r2 = 0.961; Fig. 1A).

Figure 1.

ELP tumor retention is dependent on the transition temperature. A, in vivo tumor retention of ELPs as a function of ΔTt. The ΔTt is the difference between the tumor temperature (31.7°C) in anesthetized mice and the ELP's Tt. Each point in the figure is the tumor retention (%ID/tumor) 7 days after intratumoral infusion of each [125I]ELP. B, ELP viscosity after thermal phase transition as a function of ΔTt. C, hydrodynamic radius (Rh) of ELPs at a temperature below its infusion temperature.

Figure 1.

ELP tumor retention is dependent on the transition temperature. A, in vivo tumor retention of ELPs as a function of ΔTt. The ΔTt is the difference between the tumor temperature (31.7°C) in anesthetized mice and the ELP's Tt. Each point in the figure is the tumor retention (%ID/tumor) 7 days after intratumoral infusion of each [125I]ELP. B, ELP viscosity after thermal phase transition as a function of ΔTt. C, hydrodynamic radius (Rh) of ELPs at a temperature below its infusion temperature.

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The tyrosine content also affected ELP viscosity; ELPs with greater numbers of tyrosine residues exhibited a higher viscosity than those with fewer residues (Fig. 1B). This behavior may be partially explained by the presence of 36.9 ± 12.4 nm micelles in the ELP120-Tyr7 sample. Unlike ELP120-Tyr1 and ELP120-Tyr4 samples that solely consist of soluble unimers (Rh ≈ 6 nm), the ELP120-Tyr7 population was equally distributed between a unimeric state and a nanoparticle state as seen by DLS (Fig. 1C). This suggests that when the nanoaggregates are heated to body temperature, the ELP coacervate that is formed contains a significant fraction of aggregated micelles, leading to a different internal microstructure than the other 2 ELPs, thereby affecting physicochemical properties such as its viscosity. These data are clear evidence of the importance of fine tuning the molecular architecture of ELPs and illustrate the many tools available for easily modulating intratumoral retention.

Long-lasting radiolabeled seeds lead to complete tumor regression

Tumor retention studies show that the 50 kDa ELP (ELP120) administered at 1,000 μmol/L promotes the formation of long-lasting seeds. Furthermore, increased tyrosine content was also shown to enhance tumor retention and thus the cumulative tumor exposure to radioactivity (Supplementary Table S1). Hence, to evaluate whether increased tumor retention translates into a therapeutic advantage, we investigated the tumor retention and antitumor effects of [131I]ELP120 containing 1 (ELP120-Tyr1), 4 (ELP120-Tyr4), and 7 tyrosines (ELP120-Tyr7) per molecule at an injection concentration of 1,000 μmol/L. Figure 2A shows that the temperature-triggered formation of an ELP seed containing 7 tyrosines most effectively retained the radioactive payload within the tumor mass. Correspondingly, the antitumor efficacy and survival time of the mice correlated with tumor retention, with ELP120-Tyr7 displaying the best therapeutic outcome, followed by ELP120-Tyr4 and ELP120-Tyr1 (Fig. 2B and C). The ELP120-Tyr7 seed exhibited much better antitumor efficacy than either ELP120-Tyr1 or ELP120-Tyr4 and displayed the longest median survival time (60 days vs. 39 and 17 days, respectively). In the ELP120-Tyr7 treatment group, 10/12 mice survived until the end of the study (60 days), and 8/12 tumors showed complete regression. In contrast, all of the animals in the control ELP group, 6/7 mice treated with ELP120-Tyr1, and 6/8 mice treated with ELP120-Tyr4 were euthanized before the end of the study due because of either excessive tumor size (>1 cm3; Table 1) or body weight loss (>15%) because of tumor burden. Histologic examination illustrated that this radiation damage manifested as an acute inflammatory reaction (infiltration of white blood cells and congestion of blood vessels) in the tumor tissue at 24 hours (Fig. 2E) compared with that before treatment (Fig. 2D), and the initiation of cell death at 48 hours (Fig. 2F) following administration. By 72 hours, the tumor cell outline had disappeared (Fig. 2G). However, tumor shrinkage was not observed until 5 days posttreatment (Fig. 2B).

Figure 2.

Tumor retention and antitumor efficacy of [131I]ELP in mice bearing FaDu tumor xenografts. Comparison of tumor retention of radioactivity (A), tumor growth posttreatment (B), and Kaplan–Meier plots (C) of survival of mice treated with 3 different [131I]ELPs containing 1, 4, or 7 C-terminal tyrosine residues. Data represent mean ± SEM (n = 7–12). D–F and G show HE stained tumor tissue sections obtained 0, 24, 48, and 72 hours, respectively, after intratumoral infusion of [131I]ELP120-Tyr7.

Figure 2.

Tumor retention and antitumor efficacy of [131I]ELP in mice bearing FaDu tumor xenografts. Comparison of tumor retention of radioactivity (A), tumor growth posttreatment (B), and Kaplan–Meier plots (C) of survival of mice treated with 3 different [131I]ELPs containing 1, 4, or 7 C-terminal tyrosine residues. Data represent mean ± SEM (n = 7–12). D–F and G show HE stained tumor tissue sections obtained 0, 24, 48, and 72 hours, respectively, after intratumoral infusion of [131I]ELP120-Tyr7.

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Table 1.

Comparison of tumor retention of radioactivity and antitumor efficacy of the optimized [131I]ELP-depot in FaDu and PC-3 tumor xenografts in mice

FaDuPC-3
ParameterUnlabeled ELP120-Tyr4[131I]ELP120-Tyr1[131I]ELP120-Tyr4[131I]ELP120 -Tyr7Unlabeled ELP120-Tyr7[131I]ELP120-Tyr7
Number of mice 11 12 10 13 
Cumulative activity (day·μCi/tumor) 3,597 8,636 17,086 15,694 
Half-life of radioactivity, d NA 3.2 5.27 7.08 NA 6.42 
Growth rate at day 11, mm3/d 85 17 −5 53 −1.19 
Median survival, d 17 39 60 30 60 
Complete regression 0/11 0/7 2/8 8/12 0/10 10/13 
Survived at day 60 0/11 1/7 2/8 10/12 0/10 13/13 
FaDuPC-3
ParameterUnlabeled ELP120-Tyr4[131I]ELP120-Tyr1[131I]ELP120-Tyr4[131I]ELP120 -Tyr7Unlabeled ELP120-Tyr7[131I]ELP120-Tyr7
Number of mice 11 12 10 13 
Cumulative activity (day·μCi/tumor) 3,597 8,636 17,086 15,694 
Half-life of radioactivity, d NA 3.2 5.27 7.08 NA 6.42 
Growth rate at day 11, mm3/d 85 17 −5 53 −1.19 
Median survival, d 17 39 60 30 60 
Complete regression 0/11 0/7 2/8 8/12 0/10 10/13 
Survived at day 60 0/11 1/7 2/8 10/12 0/10 13/13 

The antitumor efficacy of these ELP seeds was also examined in PC-3 human prostate carcinoma xenografts. Both the tumor retention and the antitumor efficacy results for the PC-3 xenografts (Fig. 3A–E) were comparable to those observed in the FaDu line (Table 1). In both tumor lines, [131I]ELP120-Tyr7 promoted prolonged tumor retention of 131I activity as measured by the area under the curve (cumulative radioactivity), half-life of tumor radioactivity (Table 1) and suppressed tumor growth. Moreover, this treatment significantly prolonged the survival of mice bearing PC-3, whereas 10/10 of the control mice with PC-3 tumors died within 35 days and 13/13 of the treated mice survived until the end of the 60-day study. Histologic examination of PC-3 tumors from these long-term survivors showed that the ELP seeds eliminated all of the tumor cells remaining at the tumor site (Fig. 3G and I), whereas the control ELPs permitted tumor growth and fibrosis (Fig. 3F and H). These results show that this locally infused, self-assembled seed has significant therapeutic effect against 2 different human tumor lines.

Figure 3.

Tumor retention and antitumor efficacy of [131I]ELP in mice bearing PC-3 tumor xenografts. Radioactivity retention (A), antitumor efficacy (B), and survival following intratumoral infusion of [131I]ELP120-Tyr7 (C). Data represent mean ± SEM (n = 10–13). Photographs of PC-3 tumors 23 days after treatment with unlabeled ELP (D) and 60 days after treatment with [131I]ELP120-Tyr7 (E). H&E stained tumor tissue sections obtained 23 days after treatment with unlabeled ELP (F and H) or 60 days after treatment with [131I]ELP120-Tyr7 (G and I).

Figure 3.

Tumor retention and antitumor efficacy of [131I]ELP in mice bearing PC-3 tumor xenografts. Radioactivity retention (A), antitumor efficacy (B), and survival following intratumoral infusion of [131I]ELP120-Tyr7 (C). Data represent mean ± SEM (n = 10–13). Photographs of PC-3 tumors 23 days after treatment with unlabeled ELP (D) and 60 days after treatment with [131I]ELP120-Tyr7 (E). H&E stained tumor tissue sections obtained 23 days after treatment with unlabeled ELP (F and H) or 60 days after treatment with [131I]ELP120-Tyr7 (G and I).

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The biodegradable radioactive seeds show minimal toxicity in mice

To address the concern that the biodegradability of these seeds and egress of labeled catabolites from tumor could lead to excessive radiation exposure of normal tissues, we monitored the overall health of the mice receiving therapy-level doses through body weight measurements. In addition, the biodistribution of the ELP following i.t. injection was assessed. During the course of radiotherapy, none of the animals in either tumor model exhibited significant clinical signs of toxicity The treatment of FaDu xenografts with 2,000 μCi [131I]ELP120-Tyr7, for instance, showed a maximum body weight loss of 4% (Fig. 4A); generally, a 15% reduction in body weight is accepted before the mouse must be euthanized for humane reasons. Body weight loss in the mice bearing the PC-3 xenograft, however, was more severe, as the maximum body weight loss reached a plateau of 14% on day 15, gradually improving to normal levels over the following 2 weeks (Fig. 4B). However, this toxicity was not primarily caused by the ELP seed, but rather by the PC-3 tumor. Mice bearing the PC-3 tumor displayed greater than 10% weight loss in the absence of treatment (data not shown). The biodistribution results further show that after i.t. administration of this radiotherapeutic depot, clearance of radioiodine from normal tissues was rapid, with the result that by 24 hours, activity levels were less than 1% ID/g in all tissues studied including the thyroid (Fig. 4C). Autoradiography of the tumor following [131I]ELP120-Tyr7 infusion illustrate that the 131I activity was concentrated primarily at the administration site located in the center of the tumor (Fig. 4E), and its distribution pattern matched the area of tumor necrosis seen in the histologic tumor section (Fig. 4D). These results show that the [131I]ELP120-Tyr7 radioactivity was mainly restricted to the tumor, and indicate that the systematic optimization of this local drug delivery depot significantly improved antitumor efficacy and lowered systemic toxicity.

Figure 4.

Safety evaluation and localization of the ELP depot in mice. Mean body weight following i.t. administration of unlabeled ELP or [131I]ELP120-Tyr7 in nude mice bearing FaDu xenografts (A) and PC-3 xenografts (B). Data represent the mean and SEM, n = 7–12. C, the biodistribution of radioactivity in mice bearing FaDu tumor xenografts treated with an ELP depot. Data represent the mean and SEM, n = 5–6. H&E stained photomicrograph of the tumor injection site 48 hours after i.t. administration of [131I]ELP120-Tyr7 at ×50 magnification (D) and autoradiography of the corresponding tumor injection site of D (E).

Figure 4.

Safety evaluation and localization of the ELP depot in mice. Mean body weight following i.t. administration of unlabeled ELP or [131I]ELP120-Tyr7 in nude mice bearing FaDu xenografts (A) and PC-3 xenografts (B). Data represent the mean and SEM, n = 7–12. C, the biodistribution of radioactivity in mice bearing FaDu tumor xenografts treated with an ELP depot. Data represent the mean and SEM, n = 5–6. H&E stained photomicrograph of the tumor injection site 48 hours after i.t. administration of [131I]ELP120-Tyr7 at ×50 magnification (D) and autoradiography of the corresponding tumor injection site of D (E).

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In this study, we developed an alternative modality to conventional brachytherapy consisting of a biodegradable, genetically engineered polypeptide conjugated to a therapeutic radioisotope that spontaneously transitions from an easily infusible liquid into a seed-like depot upon injection into a solid tumor. We observed that the integrity and efficacy of this depot correlated with the number of tyrosines in its C-terminus tail, overall MW, and solution concentration, and showed that these spontaneously assembled seeds are effective against 2 human tumor xenografts in athymic mice. We hypothesized that the physicochemical and structural properties of the polypeptide seeds could be altered to maximize depot retention and efficacy.

To investigate this hypothesis, we recombinantly synthesized a series of polypeptides, prepared ELP-radionuclide conjugates, and investigated the effect of polypeptide MW, amino acid composition, and injection concentration—the three main parameters controlling their phase transition behavior—on the stability of the depot. Interestingly, we found that each parameter played an important role. Increasing the injection concentration and increasing the number of tyrosines in the sequence increased tumor retention half-life. Increasing the MW also increased tumor retention, though only up to a point; doubling the MW from 50 to 100 kDa did not result in a further increase in seed stability. Importantly, the increased retention time directly correlated with the ΔTt, defined as the difference between the transition temperature of the ELP under these specific conditions and the intratumoral temperature. We hypothesized that by using these parameters to drive the absolute transition temperature lower—thereby increasing the ΔTt—we effectively increase seed stability by altering 2 molecular parameters: viscosity and internal microstructure. We have 2 lines of evidence suggesting that viscosity and the internal microstructure are important. The first is that the most successful depot, ELP120-Tyr7, also exhibits the highest viscosity (Fig. 1B). The markedly different viscoelastic behavior of this depot compared with the 2 other ELPs studied herein is probably related to the fact that its internal microstructure is quite different from the other 2 ELP depots. The second piece of evidence was revealed by DLS: ELP120-Tyr7 forms spherical micelles at temperatures below its coacervation temperature, unlike ELP120-Tyr1 and ELP120-Tyr4 (Fig. 1C). Thus, the microstructure of this ELP seed may consist of aggregated nanoscale micelles, whereas the other ELP seeds simply consist of entangled polymer chains with little internal structure. This difference in microstructure, we believe, is largely responsible for the stark difference in the temporal retention of the seed within the tumor, and hence its efficacy in both confining the radioactivity to the tumor space—thereby limiting systemic toxicity—and enhancing efficacy via its ability to irradiate the tumor from the inside out.

Given that this spontaneously formed seed was stable for prolonged periods of time, we hypothesized that it could prove an effective therapeutic for the treatment of tumors in regions accessible by injection such as the head, neck, breast, brain, and prostate. This seed-like depot displayed significant antitumor efficacy against 2 human tumor xenografts as seen by the following results: (i) 100% of the tumors responded to treatment, resulting in a 67% cure rate and (ii) 8/12 mice bearing FaDu tumors and 13/13 mice bearing PC-3 tumors survived until the end of the 60-day experiment. The tumor response was separated into 2 phases, which is typical of clinical brachytherapy. In the first stage (3–5 days posttreatment), the tumor mass was large and rigid, which was likely because of edema of the tumor tissue. In the second stage, approximately 3 to 7 days postinfusion, the tumor size decreased and the mass became more pliant. Histologic analysis indicated that this effect was likely because of necrosis because significant tumor tissue destruction in and around the injection site was observed 72 hours postinfusion of the radiolabeled ELP.

We also examined the toxicity of this system in preliminary fashion by monitoring body weight of animals receiving [131I]ELP. Similar to clinical brachytherapy, this modality was well tolerated by mice implanted with 2 different human tumor xenografts, suggesting that the therapeutic load was predominantly contained within the tumor. The biodistribution results showed that only a limited amount of the radioactivity accumulated in important organs (Fig. 4C). Noteworthy, there were 3 forms of radioactivity distributed in the tumor and in normal organs, including free 131I, free [131I]ELP, and aggregated [131I]ELP, and each one of these is expected to have a different elimination pathway. Free 131I is rapidly cleared once released from the ELP into circulation; free [131I]ELP may be cleared off at a slower rate as it diffuses away from the ELP depot and enters systemic circulation. The mechanism of clearance of these 2 forms may vary depending on its degradation rate and MW, but our biodistribution data (Fig. 4C) suggests a gradual excretion process through the kidneys, as indicated by a higher percentage of the injected dose in the kidneys compared with other organs at each time point. The toxicity contributed by this clearance process will only be minor, however, because we observed that the exposure rapidly dropped below 0.5% ID/g for all organs after the first 24 hours after administration (Fig. 4C). This also allows us to assume in our subsequent analyses that more than 99% of the detected tumor radioactivity originates from the aggregated [131I]ELP that constitutes the major radioactive source in our injectable system. These encouraging results suggest that this polypeptide seed may by worthy of further evaluation for several potential applications: (i) to treat patients who are at an early stage (e.g., breast or prostate) without requiring them to undergo radical surgery; (ii) to make unresectable tumors operable by debulking their mass (e.g., tumors near major arteries or organs); and (iii) to improve the quality of life for patients with advanced tumors (e.g., lung cancers that obstruct the bronchi).

Despite the efficacy of this procedure, we are currently investigating methods to further improve its therapeutic potential. For example, this treatment strategy relies on a uniform coverage of the entire tumor because the penetration depth of the selected radionuclide (131I) is only 1 mm. While this short penetration depth can prevent harmful radiation from affecting nearby healthy tissues, it can also leave live tumor cells at the tumor boundaries (20). To improve the uniformity of the coverage it may be possible to inject multiple seeds (similar to clinical brachytherapy) to compensate for large or multifocal tumors or modulate the distance the infusion solution travels before transitioning into a stable seed-like depot. Furthermore, providing a second infusion following the initial response may prevent the tumor reemergence that we observed in a few isolated animals.

In conclusion, we have developed a genetically engineered, thermally responsive peptide polymer that self-assembles into a biodegradable seed upon intratumoral injection. By modulating the physicochemical and structural characteristics of the polypeptide, we were able to significantly increase the retention time and the antitumor efficacy of the self-assembled seeds. The intratumoral administration of the seed-like depot led to a 100% response rate and a more than 67% cure rate in 2 xenograft models after 60 days, and showed only minimal accumulation (<1%ID/g) in vital organs. This novel self-assembled seeds are now being studied in orthotopic animal models to assess its feasibility as an alternative to brachytherapy seeds.

A. Chilkoti has a financial interest in PhaseBio Pharmaceuticals, which has licensed the rights to the local delivery technology described herein from Duke University. No potential conflicts of interest were disclosed by the other authors.

Conception and design: W. Liu, D. Asai, A. Chilkoti

Development of methodology: W. Liu, X. Li, D. Asai, A. Chilkoti

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W. Liu, J.R. McDaniel, X. Li, F.G. Quiroz, J. Schaal, J.S. Park

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W. Liu, J.R. McDaniel, X. Li, F.G. Quiroz, J. Schaal, M. Zalutsky

Writing, review, and/or revision of the manuscript: W. Liu, J.R. McDaniel, F.G. Quiroz, J. Schaal, J.S. Park, M. Zalutsky, A. Chilkoti

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W. Liu, X. Li, A. Chilkoti

Study supervision: W. Liu, J.S. Park, M. Zalutsky, A. Chilkoti

This research was supported by the NIH through grant 5R01CA138784-03 to W. Liu.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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