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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

A novel pyropheophorbide-a (PPa) derivative, Ac-sPPp, was developed in our lab for targeted photodynamic therapy (PDT) and combination therapies. Its versatile peptide moiety, high water-solubility, amphiphilicity, and micellar aggregation allow efficient coupling to targeting moieties and convenient mixing with other therapeutics. Photosensitizer immunoconjugate (PIC) targeted PDT, using Ac-sPPp conjugated to therapeutic anti-epidermal growth factor receptor (EGFR) antibody cetuximab, and PDT + chemotherapy combination treatment, using Ac-sPPp mixed with stealth liposomal doxorubicin (Doxil), were investigated as promising strategies for potentiating PDT and improving target specificity. Passively targeted PDT with Ac-sPPp only or surfactant-solubilized PPa was also investigated for comparison. The A-431 human vulvar squamous cell carcinoma, xenografted in nude mice, was chosen as a tumor model because of its high EGFR expression and sensitivity to liposomal doxorubicin in vitro. Fluorescence imaging and PDT experiments showed that Ac-sPPp formulations circulated far longer and provided superior tumor contrast and superior tumor control compared to PPa. Strong PDT vascular effects were observed by laser Doppler imaging regardless of whether Ac-sPPp was passively or actively targeted. Passively targeted Ac-sPPp PDT gave equivalent or better tumor control than PIC-targeted PDT or PDT + Doxil combination therapy, and when treatments were repeated, it also yielded the highest cure rate.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Our lab has developed a new photosensitizer (PS), Ac-sPPp, with the aim of creating a PS construct that can be easily coupled to cancer-specific targeting molecules or combined with other therapeutics for photodynamic combination therapy (see Fig. 1). Ac-sPPp is a highly water-soluble amphiphile consisting of the hydrophobic PS, pyropheophorbide-a (PPa), linked via a peptide to a short polyethylene glycol (PEG) tail. The peptide functional groups facilitate covalent conjugation onto targeting carriers (e.g. antibodies). Additionally, Ac-sPPp forms micellar aggregates, which allows even greater PS loading onto carriers through strong noncovalent amphiphilic interactions. Micellar Ac-sPPp solutions may also be used alone for passive targeting or directly mixed with other therapeutics.

image

Figure 1. Ac-sPPp and its combination with potentiating therapeutics. (a) Chemical structure of Ac-sPPp [sequence: N-ε-(PPa-Asp)-Lys-PEG(11-mer)-PEG(11-mer); R is an acetyl group, Ac]. Ac-sPPp was used alone as a single agent or in combination formulations with other therapeutics. (b) Ac-sPPp formulated as a PIC. PIC preparations consisted of Ac-sPPp tightly bound to an anti-erbB therapeutic antibody (cetuximab or trastuzumab) through active ester-mediated covalent linkage and strong noncovalent micellar interactions. (c) PS-chemotherapeutic mixture of Ac-sPPp with Doxil (doxorubicin encapsulated in a PEG-coated stealth liposome).

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In this study, we generated an anti-erbB PS immunoconjugate (PIC) by conjugating Ac-sPPp onto the therapeutic antibody, cetuximab, for epidermal growth factor receptor (EGFR)-targeted photodynamic therapy (PDT). We also mixed Ac-sPPp with stealth liposome encapsulated doxorubicin, Doxil, for PDT + chemotherapy combination treatment. Both approaches are considered promising strategies for potentiating PDT and improving target specificity. Anti-erbB PICs have been of interest for some time because in vitro studies have shown that they exert erbB inhibitory activity[1-3] and allow target-specific PS delivery for PDT of erbB-overexpressing cancers.[3-6] Similarly, there has been interest in PDT + Doxil combination therapy because PDT and Doxil have been shown to potentiate each other without increasing systemic toxicity or local collateral damage.[7] Liposomal doxorubicin has also been shown to be effective against erbB-overexpressing cancers.[8, 9]

Despite increasing interest in these sorts of approaches for potentiating PDT and improving target specificity, in vivo studies have been relatively limited. While there have been a large number of in vitro studies demonstrating the feasibility of PIC-targeted PDT, there have only been a handful of in vivo studies, and many confounding issues remain unanswered or inadequately addressed.[10] In particular, the hydrophobicity of many PSs and their aggregation in aqueous solutions have made it difficult to produce optimal PICs.[4, 11] Moreover, no in vivo studies to date have definitively demonstrated that PICs offer a decisive advantage over passively targeted PSs. There are similar gaps in knowledge regarding the efficacy of combining PDT with doxorubicin chemotherapy. In vitro studies suggest PDT can potentiate doxorubicin activity through a photochemical internalization effect,[12] and early pioneering in vivo studies have shown that PDT + Doxil treatment enhances doxorubicin accumulation in tumors, significantly potentiates tumor control, and increases cure rates compared to PDT only or Doxil monotherapy.[7] More recent studies have verified that PDT improves liposomal doxorubicin delivery in different tumor models, but delivery paralleled tumor vascularity, and no evidence of therapeutic potentiation was shown.[13, 14] Consequently, it is still unclear whether PDT + Doxil treatment can be broadly effective against a range of tumor types.

Another important issue is whether potentiating PDT with targeted PS formulations and combination therapies provides significant advantages compared to more basic approaches such as increasing PDT dosage or repeating PDT. Generally, there are limits to increasing PDT dosage because excessive local tissue damage may be unacceptable, and in the case of small animals, systemic toxicity and acute lethality may result due to traumatic shock syndrome mediated by endogenous vasoactive agents[15] (in initial mouse PDT experiments, we observed systemic toxicity symptoms for Ac-sPPp doses >4.5 nmole g−1 body weight using 100 J cm−2 light delivered 16 h post-PS injection). Therefore, it is usually more practical to fractionate dose and repeat PDT as needed.

In our present study of the EGFR-overexpressing A-431 human vulvar squamous cell carcinoma, which shows exceptional radioresistance[16] and exhibits high recurrence in immunocompromised mouse models[2, 17], we found that passively targeted Ac-sPPp PDT provides superior tumor control compared to PPa PDT and equivalent or better tumor control compared to anti-EGFR Ac-sPPp-cetuximab PIC-targeted PDT or Ac-sPPp + Doxil PDT combination therapy. We also observed strong vascular PDT effects, regardless of whether Ac-sPPp was targeted passively as a micellar solution or actively as a PIC. Finally, we found that repeating PDT offers a higher cure rate than potentiating treatment with anti-EGFR PIC-targeted PDT or PDT + Doxil combination therapy.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Cell lines

Human A-431 squamous cell carcinoma and SK-BR-3 breast cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cell culture passaging was always limited to less than 6 months after resuscitating frozen cell stocks. ATCC rigorously authenticates its cell lines using standardized methods described in its cell biology program documentation (www.atcc.org). An optically reporting A-431/G4 clonal cell line expressing a red fluorescent protein and luciferase was developed using lentiviral methods.[17] Cell line maintenance conditions have previously been described elsewhere.[17]

Antibodies

Cetuximab (anti-EGFR/erbB1) and trastuzumab (anti-HER2/erbB2) were obtained from ImClone Systems Incorporated (Branchburg, NJ) and Genentech, Inc. (South San Francisco, CA), respectively.

Photosensitizer and combination formulations with potentiating therapeutics

Synthesis and characterization of Ac-sPPp (see Fig. 1a) has previously been described.[17] Dynamic light scattering (DLS) was used to measure particle size distributions of Ac-sPPp solutions (Zetasizer Nano ZS; Malvern Instruments, Inc., Southborough, MA).

Ac-sPPp PICs were prepared by activating Ac-sPPp as an N-hydroxysuccinimide (NHS) ester and then reacting it with antibodies. To obtain the Ac-sPPp-NHS active ester, 0.9 μmole of Ac-sPPp dissolved in 0.3 mL of methylene chloride was mixed with 2 μL of triethylamine and 10 mg of di(N-succinimidyl) carbonate (DSC; Sigma-Aldrich Corporation, St. Louis, MO)[18] by vigorous vortexing for 5 min. Excess DSC was then removed by centrifugation, and the supernatant was collected and divided into aliquots of ∼100–200 nmole Ac-sPPp-NHS. Solvent was evaporated from the aliquots using a freeze dryer concentrator (Savant SPD 131 DDA; Thermo Electron Corporation, Milford, MA). To generate PICs, Ac-sPPp-NHS aliquots were dissolved in dimethyl sulfoxide (DMSO) just prior to reaction with antibodies. Typically, one aliquot of Ac-sPPp-NHS was used to label 0.5 mg of antibody. Procedures for preparing Ac-sPPp PICs were essentially the same as previously published protocols for preparing PPa and verteporfin PICs, except PEGylation of the antibody prior to labeling with PS was not required.[4, 6] Pre-PEGylation of the antibody was unnecessary because Ac-sPPp already contains a PEG moiety, which helps prevent aggregation and maintain PIC solubility without requiring any additional PEGylation. UV–Visible absorbance spectroscopy and SDS-PAGE methods, described in previous studies, were used to assess PIC molar labeling ratios and to estimate the amount of noncovalently associated PS in the PIC preparations.[4, 6]

Photosensitizer-chemotherapeutic mixtures were prepared by vortexing Ac-sPPp and Doxil (2 mg mL−1 doxorubicin HCl encapsulated in a PEG-coated stealth liposome; Ben Venue Laboratories, Inc., Bedford, OH) solutions together until they were homogeneously mixed. Mixtures contained ∼55–75 nmole Ac-sPPp in Dulbecco's phosphate buffered saline without Ca2+ or Mg2+ (PBS) and ∼135–190 μg doxorubicin equivalents of Doxil in its original excipient solution (200 μL total mixture volume). To determine if Ac-sPPp + Doxil mixtures remained stable after overnight incubation at 37°C, samples were ultracentrifuged (average RCF = 292 × 103 g for ≥1 h at 20°C; Optima MAX-E Ultracentrifuge, TLA-100 rotor; Beckman Coulter, Inc., Fullerton, CA), and sediments were examined by visual inspection and absorbance spectroscopy.

In vitro phototoxicity

Phototoxicity studies of cultured cells were conducted using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. PS stock solutions were 80 to 90 μm and were diluted into culture media with rapid mixing to attain desired concentrations (typically 50–300 nm PPa equivalents). Ac-sPPp and Ac-sPPp PIC stock solutions were prepared in PBS, but PPa was prepared as a DMSO stock solution since it is not directly soluble in aqueous solutions. Details of the MTT-based assay have previously been described elsewhere.[6] Alternatively, for optically reporting cells, viability was more easily assessed using luciferase bioluminescence. MTT and bioluminescence viability assays were found to give similar results in control studies. For the luciferase-based phototoxicity assay, all treatment conditions were similar to the MTT-based assay, except cells were grown and PDT treated in 96-well black-welled/clear-bottom plates. Viability was assayed 12–24 h post-PDT by incubating cells with 0.15 mg mL−1 luciferin in complete culture medium for 5 min at 37°C and then immediately imaging plates on a custom-built optical imager.[17] Wells containing untreated cells served as control comparisons.

Animals and tumor models

Female athymic NCr-nu/nu mice, 4–5 weeks old, were obtained from the Animal Production Program of the National Cancer Institute (Frederick, MD). A-431 or optically reporting A-431/G4 tumors were implanted subcutaneously or intradermally in the pectoral or pelvic regions for imaging and PDT experiments. Tumor inoculation, tumor measurement, and anesthesia methods have been previously described.[17] All animal experimentation was approved by Dartmouth College's Institutional Animal Care and Use Committee.

In vivo PS fluorescence imaging

Mice bearing A-431 tumors 100–200 mm3 in size were administered various PS formulations via lateral tail vein injection. A home-built optical imager was used to take fluorescence images of the mice pre-injection and at various time points post-injection for monitoring PS clearance and temporal changes in biodistributions. Mice were anesthetized with a ketamine/xylazine cocktail for imaging. Detailed descriptions of the optical imager, imaging and anesthesia procedures, and image processing and analysis methods have been reported elsewhere.[17] For injections, Ac-sPPp and Ac-sPPp PICs were prepared in PBS, while PPa was prepared in a 1% polyoxyethylene sorbitan monooleate (Tween 80)/2% ethanol/∼0.2 mm Na2CO3/D5W (5% dextrose in water) surfactant solution,[19, 20] due to its insolubility in water. Total PS injection volume per mouse was 200 μL.

In vivo PDT

Mice bearing A-431 tumors were administered various PS formulations using the same procedures as described for fluorescence imaging. For Ac-sPPp PDT + Doxil chemotherapy experiments, a pre-mixed solution of Ac-sPPp and Doxil (3 nmole Ac-sPPp and 7.5 μg doxorubicin HCl equivalents per g body weight) was administered. Sixteen or forty hours post-PS injection, mice were anesthetized with ketamine/xylazine, and red light (670 nm, ∼50 mW cm−2, 100 J cm−2) was applied to the tumor area, which included a ∼2 mm margin of normal tissue around the tumor perimeter. Control mice were given no PDT (no PS and no light) or Doxil only with no PDT (7.5 μg doxorubicin HCl equivalents per g body weight). Tumors were ∼150 mm3 at the time of treatment initiation. Details of the PDT illumination setup and irradiation procedures have been described elsewhere.[17] After PDT, mice that had not yet awakened from anesthesia were kept warm in a 37°C incubator and then returned to cages upon recovery. For a subset of mice, PDT was repeated when tumors had regrown to a volume equal to or slightly smaller than the original pre-PDT size (∼150 mm3). An unpaired one-tailed t-test for unequal variances was used to assess whether differences in post-treatment tumor growth times between two different treatment groups were statistically significant (< 0.05). For the purposes of this analysis, post-treatment growth time was defined as the time required for tumor volume to reach 400 mm3 following initiation of treatment.

Monitoring PDT treatment effects

Mice were photographed and imaged by various methods to document and quantify a variety of PDT effects. For all imaging procedures, mice were anesthetized in a similar manner as described above for fluorescence imaging and PDT experiments. Changes in gross appearance of tumors and normal surrounding tissues were recorded by digital photography (DSC-T2 camera; Sony Corp., Tokyo, Japan). Changes in tumor bioluminescence and PS fluorescence photobleaching were monitored by optical imaging using a home-built imager (imaging setup and procedures for optical imaging have been described elsewhere).[17] Scanning laser Doppler imaging (LDI) was used to assess PDT-induced changes in tissue perfusion (moorLDI imager; Moor Instruments Inc., Wilmington, DE). For LDI experiments, tumors were grown superficially in the intradermal layer of the left pelvic region to maximize Doppler signal originating from tumor tissue and to minimize noise from respiratory movement. Except for a small opening for laser scanning access, mice were fully enclosed in a 37°C water-jacketed box during LDI to maintain stable temperature and limit tissue perfusion perturbations caused by temperature variation.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Ac-sPPp and its combination formulations with potentiating therapeutics

Ac-sPPp (see Fig. 1a) and its hydrophobic parent compound PPa have similar photophysical characteristics (e.g. their absorbance spectra under aggregating and disaggregating conditions are nearly identical; data not shown), but Ac-sPPp is highly water-soluble and dissolves directly in aqueous solutions without requiring surfactants. DLS experiments indicate that Ac-sPPp forms irregular aggregates of a wide range of sizes in purely aqueous solutions. Conversely, aqueous Ac-sPPp mixed with the serum protein albumin is highly monodisperse with particle sizes similar to the protein, suggesting Ac-sPPp probably forms micellar aggregates that nucleate around and adsorb onto proteins.

In an attempt to enhance PS tumor targeting and improve PDT efficacy, PICs were prepared by reacting an active NHS ester of Ac-sPPp with anti-erbB therapeutic antibodies. Stringent gel filtration with a DMSO/aqueous eluant, which allowed preparation of relatively high purity PPa and verteporfin PICs in past studies (containing <10% noncovalently associated PS),[4, 6] helped remove unbound Ac-sPPp from the PICs. However, SDS-PAGE indicated that Ac-sPPp PICs still contained appreciable amounts of noncovalently associated Ac-sPPp, ranging as high as 42%. PIC molar labeling ratios, calculated from absorbance measurements, were ∼11 to 14 Ac-sPPp molecules per antibody. This means ∼6 to 8 Ac-sPPp molecules were bound by covalent linkage, and ∼5 to 6 Ac-sPPp molecules were tightly bound via noncovalent micellar interactions (see Fig. 1b). As with Ac-sPPp, PICs were highly water-soluble and could be sterile filtered through 0.2-μm membranes with minimal losses. No insoluble aggregates formed even after a year of storage at 4°C.

Mixtures of Ac-sPPp and Doxil were prepared with the aim of potentiating PDT with chemotherapy while only requiring a single-injection administration (see Fig. 1c). To make Ac-sPPp + Doxil mixtures, a stock solution of Ac-sPPp in PBS was thoroughly mixed with Doxil. Stability of mixtures after overnight incubation at 37°C was investigated by ultracentrifugation. Ac-sPPp did not incorporate into Doxil liposomes (no significant amount of Ac-sPPp was observed in the ultracentrifuged Doxil sediment layer), and Doxil liposomes remained intact with no leakage of doxorubicin (no significant amount of free doxorubicin was seen in supernatants).

In vitro phototoxicity experiments

Studies were conducted to compare the in vitro phototoxicity and targeting specificity of various PS formulations. Representative in vitro phototoxicity data are provided in the Supplementary Materials (see Fig. S1). In general, PPa was found to be significantly more phototoxic than Ac-sPPp, and compared to anti-erbB Ac-sPPp PICs, PPa and Ac-sPPp exhibited no targeting specificity in vitro. Phototoxic killing of A-431/G4 cells was >95% for 150 nm PPa but was negligible for 150 nm Ac-sPPp, and >95% killing was only achieved with Ac-sPPp above ∼500 nm (10 J cm−2 light dose, 16- or 40-h incubations). By comparison, 150 nm PPa equivalents of anti-EGFR Ac-sPPp-cetuximab PIC with 10 J cm−2 light killed 84% and 89% of A-431/G4 cells for 16- and 40-h incubations, respectively, and phototoxic effects were inhibited to <4% killing when the PIC was co-incubated with four times the equivalent concentration of competing native cetuximab. Moreover, similar PDT treatments with 150 nm PPa equivalents of an anti-HER2/erbB2 Ac-sPPp-trastuzumab PIC resulted in negligible killing of A-431/G4 cells. Considering that A-431 and A-431/G4 cells express very high EGFR levels (∼2.5 × 106 receptors per cell) but only low HER2 levels (<10% the level of the HER2/erbB2-overexpressing cell line SK-BR-3, which expresses ∼2 × 106 erbB2 receptors per cell),[17, 21-23] the Ac-sPPp PICs exhibited highly specific targeting under the specified in vitro conditions. In fact, these results are comparable to those we have previously reported using PPa- and verteporfin-based PICs, which contained lower amounts of noncovalently associated PS (<10%).[4, 6]

However, PIC targeting was not as specific or was even nonspecific when non-target low EGFR-expressing SK-BR-3 cells (EGFR levels ∼1% that of A-431s)[22] were PDT treated with anti-EGFR Ac-sPPp-cetuximab PIC as a control, or when PIC and light doses were increased above certain levels. Supporting data are provided in the Supplementary Materials (Figs. S1c, d). For example, phototoxic killing of SK-BR-3 cells was ≥90% for 150 nm PPa and was only 40–50% for 150 nm PPa equivalents of Ac-sPPp-cetuximab PIC (10–40 J cm−2 light, 40-h incubation), but this moderate PIC phototoxicity towards SK-BR-3 cells appears to have been at least partially nonspecific given that it was only minimally inhibited by competition with four-fold equivalents of native cetuximab. Additionally, killing of A-431/G4 cells was >95% for high dose treatments using ≥300 nm PPa equivalents of anti-erbB Ac-sPPp PICs with light doses ≥20 J cm−2, and these treatments likewise were only slightly inhibited by competition with native anti-erbB antibodies.

Fluorescence time-course imaging

Representative data from fluorescence time-course imaging studies comparing A-431 tumor-bearing mice given PPa in a 1% Tween80/2% ethanol surfactant solution or Ac-sPPp in PBS are shown in Fig. 2. The images and corresponding region of interest (ROI) data show that PPa clears very rapidly from the circulation in comparison to Ac-sPPp. PPa tumor fluorescence peaked almost immediately after injection with most of the PPa dose being quickly eliminated via hepatobiliary clearance. In contrast, Ac-sPPp tumor fluorescence peaked around 6 h post-injection and was also eliminated predominantly through hepatobiliary clearance but at a much slower rate. The much longer circulation time of Ac-sPPp resulted in far superior tumor fluorescence contrast compared to PPa. Moreover, the somewhat faster clearance of Ac-sPPp from normal tissues than from the tumor resulted in a gradual increase in Ac-sPPp tumor contrast, which peaked around 24 h post-injection.

image

Figure 2. Fluorescence time-course imaging comparing Ac-sPPp with the parent PS, PPa. Mice bearing A-431 tumor xenografts implanted subcutaneously in the left pectoral region were injected via the tail vein with Ac-sPPp solubilized in PBS or with PPa prepared in a 1% Tween 80/2% ethanol surfactant solution (tumor size ∼100–200 mm3 at the time of injection). The injected PS dose was ∼3 nmol PPa equivalents per g body weight. Mice were repeatedly imaged over time to monitor changes in PS fluorescence. Images are of the ventral surface. Region of interest (ROI) analysis was performed comparing the tumor with adjacent normal chest to assess tumor fluorescence contrast over time (charted data shown below the images). Similar time-course imaging data for Ac-sPPp-cetuximab PIC is provided in the Supplementary Materials, Fig. S2.

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Fluorescence time-course imaging was also done with Ac-sPPp PICs and Ac-sPPp + Doxil mixtures. Representative data from Ac-sPPp-cextuximab PIC imaging experiments are provided in the Supplementary Materials (see Fig. S2). PIC imaging was similar in many ways to Ac-sPPp imaging, but PIC circulation times were notably longer and clearance rates were slower with residual fluorescence persisting significantly longer in the liver and tumor. Overall PIC fluorescence was also somewhat less than that of Ac-sPPp up to ∼16 h post-injection, most likely because conjugation partially quenches PS fluorescence.[4] PIC tumor fluorescence peaked around 6 h post-injection, and PIC tumor contrast peaked around 40 h post-injection. Imaging results obtained with Ac-sPPp + Doxil mixtures (3 nmole Ac-sPPp and 7.5 μg doxorubicin equivalents per g body weight) were essentially the same as those for Ac-sPPp given as a single agent, indicating that the pharmacokinetics and biodistribution of Ac-sPPp was not appreciably affected by mixing with Doxil (data not show).

In vivo PDT experiments

A-431 tumor-bearing mice were treated with different PDT regimens, and response to therapy was monitored by tumor volume measurements (see Fig. 3). PPa PDT or control treatments with Doxil only (no PDT) had negligible antitumor effects (Fig. 3a and d), while Ac-sPPp PDT, Ac-sPPp-cetuximab PIC PDT, and Ac-sPPp + Doxil PDT treatments resulted in notable tumor regressions and substantially delayed tumor regrowth (Fig. 3b–d). However, when given as single treatments, the various PDT regimens very rarely resulted in complete tumor eradication. In an attempt to eradicate tumors, Ac-sPPp PDT and Ac-sPPp-cetuximab PIC PDT treatments were given as repeated regimens (Fig. 3e and f). Repeated Ac-sPPp PDT resulted in tumor eradication in all mice, but repeated Ac-sPPp-cetuximab PIC PDT resulted in tumor eradication in only one of four mice. Overall, Ac-sPPp PDT was more efficacious than Ac-sPPp-cetuximab PIC PDT. Using post-treatment tumor growth times and number of cures as metrics for comparisons, Table 1 provides a summary of the tumor growth response data presented in Fig. 3 and indicates where differences between the various treatment groups were found to be statistically significant.

Table 1. Summary of in vivo PDT data presented in Fig. 3Thumbnail image of
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Figure 3. Tumor growth response of A-431 tumor-bearing mice PDT treated with various PS formulations. Mice were treated with PDT when tumors reached ∼150 mm3 (corresponding to the dashed vertical line at 0 days). For PDT, PS formulations were administered at a dose of ∼3 nmol PPa equivalents per g body weight followed by a 100 J cm−2 red light dose (670 nm, ∼50 mW cm−2) applied to the tumor area 16 h later. PS formulations included free PPa solubilized in 1% Tween 80/2% ethanol (a, open squares), Ac-sPPp (b and e, open triangles), Ac-sPPp-cetuximab PIC (c and f, filled triangles), and Ac-sPPp + Doxil (d, filled squares). Mice received either a single PDT treatment (a–d) or two PDT treatments (e and f, arrows indicate the timing of the second treatment). In the graphs, each tumor growth curve represents a different mouse. Number of mice treated with each regimen is indicated by n. For comparison, each graph shows tumor growth curves for a group of untreated control mice (no PS and no light, gray open circles; = 10) along with an average exponential fit of these control data (thick line). Control mice treated with Doxil only are also shown (d, filled circles). The dosage of Doxil was 7.5 μg doxorubicin equivalents per g body weight.

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In most cases, PDT-treated mice exhibited a small temporary weight loss (usually <5–10% of pre-PDT body weight) and a subsequent short delay in normal weight gain. This was probably due to the time required to recover from the PDT procedure and any post-PDT sequelae, including recovery from anesthesia and treatment-induced tissue swelling and edema that lasted ∼24 to 36 h post-PDT. However, mice treated with Ac-sPPp + Doxil PDT suffered more pronounced temporary weight loss (up to ∼15% pre-PDT body weight) than mice treated with other PDT regimens.

Tissue response to PDT

Fig. 4a shows representative photographs of a mouse treated with curative repeated Ac-sPPp PDT, and Fig. 4b shows the corresponding tumor bioluminescence signal over the course of treatment. Notable features are striking blanching and early swelling and edema in the irradiated area immediately post-PDT with eschar development and subsequent wound healing over the following 2 to 3 weeks. Following the first PDT treatment, tumor bioluminescence dropped dramatically, but tumor regrowth with an accompanying rebound in bioluminescence began around 5 days later. A second PDT treatment 3 weeks after the first treatment brought tumor bioluminescence below detectable levels, indicating complete tumor eradication (no recurrence observed out to >130 days post-PDT). Although varying somewhat in degree, the sorts of gross tissue responses observed for the various PDT regimens examined in this study were generally very similar to those depicted in Fig. 4 (except PPa PDT, which had negligible effect). Following wound healing, PDT damaged skin in and around the treated area typically contracted slightly and in some cases resulted in a small reduction in the adjacent forelimb's range of motion. In addition, nipples near the treatment area were sometimes damaged, slightly displaced, or missing altogether, and there was often skin scarring that appeared as darker and/or thinner-skinned areas.

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Figure 4. Tumor and normal tissue response during and after curative repeat treatment with Ac-sPPp PDT. Treatment parameters were the same as described in Fig. 3. Ac-sPPp was administered on days 0 and 21, and PDT irradiations were performed 16 h later on days 1 and 22. (a) Representative photographs of an A-431/G4 tumor-bearing mouse at various time points showing PDT damage of the tumor and surrounding skin area with subsequent healing and complete elimination of the tumor. (b) Corresponding tumor bioluminescence signal over the course of treatment. The dashed line indicates the estimated signal level below which tumor could no longer be definitively detected. Arrows indicate when PDT was administered.

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PDT-induced perfusion shutdown

Laser Doppler imaging experiments demonstrating PDT-induced changes in tissue perfusion are shown in Fig. 5. Tumor perfusion was found to be dramatically reduced immediately following Ac-sPPp PDT, but was unaffected by mock PDT (light treatment without any PS). Conversely, unirradiated contralateral normal tissue areas in PDT-treated mice showed no marked changes in perfusion after treatment. Perfusion of tumors before PDT was always significantly greater than perfusion of contralateral normal tissue areas. After PDT, tumor perfusion typically dropped slightly below that of the contralateral normal tissue and never recovered. Similar LDI results were obtained with Ac-sPPp PIC PDT, although the drop in tumor perfusion was not always as immediate and took up to ∼1 h post-treatment to reach a comparable degree of shutdown (data not shown).

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Figure 5. Scanning laser Doppler imaging (LDI) showing changes in tissue perfusion before and after PDT. Mice bearing A-431 tumor xenografts implanted intradermally in the left pelvic region were treated with Ac-sPPp PDT or mock PDT when tumors reached ∼150 mm3. Except for a slightly lower PS dose of ∼2.5 nmol PPa equivalents per g body weight, the Ac-sPPp PDT treatment parameters were the same as described in Fig. 3. For mock PDT, no PS was given but the light dose was the same as for Ac-sPPp PDT. (a) LDI images of the lower abdomen and pelvic region approximately 5 min before and 30 min after PDT. In each image, the circled regions of interest demarcate tumor (T) and contralateral normal tissue (NT) areas. (b) Tumor and normal tissue ROI perfusion versus time for mice shown in (a). Mice were PDT irradiated during the 0 to ∼33 min interval (indicated by the dashed vertical lines).

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PS fluorescence changes before and after PDT

Representative data showing pre-PDT temporal changes in PS fluorescence biodistribution and photobleaching immediately post-PDT are shown in Fig. 6. Only data for a mouse treated with Ac-sPPp PDT are shown since results were qualitatively similar for other PDT regimens. In general, immediately after PS injection, a significant fraction of PS was still within the major vessels. By 16 h post-injection, the PS had more evenly distributed systemically with notably higher PS retention in the tumor. Immediately after PDT, photobleaching was greatest at the tumor site, but there was also extensive photobleaching throughout the animal. Percentage decrease in ROI fluorescence immediately after PDT compared to immediately before was 86% for the tumor and 42–73% for other non-tumor tissues (see Fig. 6b). PDT photobleaching depleted a large fraction of actively circulating PS. However, fluorescence levels did not approach pre-PS-administration baseline levels until ≥5 days post-PDT.

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Figure 6. Fluorescence imaging before and after PDT. (a) Ventral surface images at various time points before and after Ac-sPPp PDT of a mouse bearing an A-431 tumor xenograft implanted subcutaneously in the left pectoral region. PDT was administered when tumor volume reached ∼150 mm3 (treatment parameters were the same as described in Fig. 3). Images are representative of results obtained in numerous PDT fluorescence imaging experiments conducted over the course of the study. (b) Mean fluorescence values at various time points of different ROIs of the mouse shown in (a). Numbers over each ROI bar set indicate the percentage decrease in ROI fluorescence immediately after PDT compared to immediately before PDT. The systemic decrease in fluorescence immediately after PDT is largely due to PS photobleaching.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The new PS construct Ac-sPPp demonstrates optimal characteristics for targeted PDT and PDT combination therapies. Compared to other PSs that have been studied for these purposes, key advantages of Ac-sPPp include its conjugatable peptide functional groups, anti-fouling PEG moiety, high water-solubility, amphiphilicity, and propensity to form micellar aggregates. We have shown that Ac-sPPp can be conjugated to cancer-targeting antibodies or directly mixed with the chemotherapeutic Doxil without adversely affecting therapeutic functionalities. We have also demonstrated that Ac-sPPp by itself is highly effective for tumor fluorescence imaging and PDT, presumably due to its efficient passive targeting of the tumor-characteristic enhanced permeability and retention effect.[24, 25]

We observed highly specific in vitro PDT targeting of EGFR-overexpressing A-431 cells with an anti-EGFR Ac-sPPp-cetuximab PIC, despite the fact that the PICs used in this study contained relatively high amounts of noncovalently associated PS. This indicates that noncovalently associated Ac-sPPp in the PICs was so tightly bound via strong micellar aggregative interactions that it did not significantly leach off the PICs and cause nonspecific effects. However, under certain conditions, Ac-sPPp PICs did exhibit a degree of nonspecificity in vitro, such as mild killing of non-target SK-BR-3 cells as well as increased nonspecific phototoxicity when PIC and light doses were raised above certain thresholds. These nonspecific effects could be partly due to small amounts of Ac-sPPp coming off the PICs, but based on competition experiments, it seems likely that the PICs were also taken up nonspecifically by adsorptive pinocytosis and phagocytosis to some extent. Nonetheless, Ac-sPPp PICs still exhibited excellent specificity in vitro compared to PPa or free non-conjugated Ac-sPPp, which respectively were highly phototoxic or relatively poorly phototoxic to both target and non-target cell lines.

In vitro PDT results do not always directly translate to an in vivo setting. Whereas Ac-sPPp PDT and Ac-sPPp-cetuximab PDT provided marked regressions and prolonged control of A-431 tumor xenografts, PPa PDT had negligible effect. Differences in PS clearance rates largely explain these results. Fluorescence imaging showed that PPa, administered in a surfactant solution similar to that used for solubilizing a related hydrophobic photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl PPa (HPPH),[19, 20] was very rapidly eliminated through hepatobiliary clearance, while Ac-sPPp and Ac-sPPp-cetuximab PIC circulated far longer, allowing substantially greater PS accumulation in tumor tissue. Even though Ac-sPPp and Ac-sPPp-cetuximab tumor contrast did not actually peak until 24–48 h post-PS administration, we ultimately decided to deliver PDT light doses at 16 h based on PS dosage (3 nmol g−1 body weight) and the need to irradiate when PS tissue concentrations were still high enough to achieve potent PDT responses. One could argue that PPa PDT would have been more effective if light had instead been delivered shortly after PS administration, but it must be kept in mind that PPa tumor contrast even at early time points was always inferior to Ac-sPPp or Ac-sPPp-cetuximab tumor contrast.

Although significant tumor control was achieved after a single treatment of Ac-sPPp PDT or Ac-sPPp-cetuximab PDT, A-431 xenografts always recurred. Consequently, we reasoned that combining PDT with a chemotherapeutic such as Doxil might potentiate PDT and achieve cures after a single treatment. However, in contrast to prior work by others,[7] we did not observe any striking potentiation when PDT was combined with Doxil. Moreover, Ac-sPPp PDT + Doxil therapy caused significant temporary body weight loss. It is possible that the A-431 model may be more resistant to doxorubicin and/or PDT than the tumor model used in the pioneering PDT + Doxil study by Snyder et al.[7] In fact, control Doxil monotherapy had little or no effect on A-431 tumors. Another reason we may not have observed potentiation is that we administered Doxil mixed with the PS, whereas prior studies have given Doxil after PDT.[7, 13, 14] However, we do not believe the timing of Doxil administration could have made much difference here, since the half-life of Doxil is ∼20 h in mice.[26]

Previous targeted PDT studies have shown that treatment repetition helps achieve complete tumor eradication.[27, 28] We found that repetition of Ac-sPPp-cetuximab PDT significantly enhanced tumor control but did not consistently eradicate all tumors. However, repetition of Ac-sPPp PDT reliably eradicated all A-431 tumors (see Fig. 3e, f and Table 1). The greater efficacy of Ac-sPPp PDT compared to Ac-sPPp-cetuximab PDT may be due to differences in PS tumor penetration (related to PS size) and/or heterogeneous EGFR expression within the tumor. In view of these possibilities, we have subsequently performed studies on a limited number of mice to determine whether increasing PS dosage to 5 nmol g−1 body weight and extending the interval between PS administration and light delivery to 40 h might improve the efficacy of Ac-sPPp-cetuximab PDT by permitting deeper PIC penetration into tumor tissues and greater EGFR-mediated uptake of the PIC (see Supplementary Materials, Fig. S3). Under these conditions, repetitive Ac-sPPp-cetuximab PDT resulted in significant temporary tumor regressions but tumor control was inferior to the original Ac-sPPp-cetuximab PDT protocol (where PS dose was 3 nmol g−1 body weight, and the interval between PS administration and light delivery was 16 h). This result was remarkable given that tumor fluorescence at 40 h post-PS administration of 5 nmol PPa equivalents per g body weight of Ac-sPPp-cetuximab was actually comparable to that of 3 nmol g−1 body weight of Ac-sPPp at 16 h post-PS administration (i.e. the corresponding Ac-sPPp dosage that permitted reliable tumor eradication). It is also noteworthy considering repetitive Ac-sPPp PDT with a PS dose of 5 nmol g−1 body weight and light delivered 40 h post-PS administration gave only a minimal PDT effect (see Supplementary Materials, Fig. S3a).

Extending the interval between PS and light administration from 16 to 40 h did not improve the potency of Ac-sPPp-cetuximab PDT, but it did improve targeting specificity and prevented collateral damage to normal surrounding tissues (e.g. scabbing of normal skin around the tumor was not observed; see Fig. S3b). Improved specificity may have been due to the fact that nonspecific vascular damage was reduced, since extending the interval between PS and light administration allows more time for the PIC to clear from the circulation. Indeed, LDI showed that both Ac-sPPp PDT and Ac-sPPp-cetuximab PDT induced potent perfusion shutdown when PDT was performed only 16 h post-PS administration. Moreover, fluorescence imaging confirmed that PS photobleaching after PDT occurred not only at the tumor site but throughout the entire animal, indicating significant fractions of PS still circulated in the vasculature during PDT with either Ac-sPPp or Ac-sPPp-cetuximab. In other control experiments, we have also observed that PDT with anti-HER2 Ac-sPPp-trastuzumab PIC using a 16-h interval between PIC and light administrations induces potent PDT effects in the A-431 tumor model (data not shown). As A-431 cells express only low levels of HER2, PDT with Ac-sPPp-trastuzumab PIC would not be expected to have much effect in the A-431 tumor model if PIC treatment were strictly mediated by specific targeted binding. These results further indicate that PICs can have strong nonspecific PDT vascular effects.

A major concern when implementing targeted PDT may be striking a balance or compromise between the intended target-specific PDT effect and nonspecific PDT vascular effects, which may or may not be beneficial for a given application. This is because many targeted PS formulations currently under consideration are delivered via the vasculature, and even if they can be delivered by another route, they may still circulate in the vasculature to some degree as they clear from the body. We have seen that the vascular effects of a targeted PS can be quite significant, even if the intended target is not the vasculature. This is a critical issue as the vasculature can be one of the most sensitive targets of PDT.[29, 30] Although extending the time between PS and light administrations improved PIC specificity, better specificity came at the cost of overall reduced efficacy. Putting these results in perspective, it will be necessary to judge all targeted PDT studies more critically, since many studies may have underestimated or overlooked nonspecific vascular effects.[10, 28, 31]

The question remains whether a passively targeted PS may be better in the end, at least for some applications, since we have shown herein that repeated Ac-sPPp PDT provides the most reliable eradication of recurrent A-431 tumors. In ongoing work, we are finding that repeated Ac-sPPp PDT can also be quite effective for controlling other tumors. For example, we have observed that Ac-sPPp PDT can provide prolonged primary tumor control in an aggressive MDA-MB-231/LM2 breast cancer xenograft mouse model,[32] although all PDT-treated mice ultimately succumbed to metastatic disease. These latest findings will be presented in detail in a subsequent publication.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by a nanocancer pilot grant from the Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center and by the National Cancer Institute through K01 CA 109567 (M.D.S.; the NIH/NCI specify that the research content is solely the authors' responsibility and does not necessarily represent official views of the NIH/NCI). Additional support was provided by Dartmouth College's Undergraduate Research Fellowship programs (E.E. Just Fellowship—N.O.; Howard Hughes Medical Institute Fellowships—J.S. and T.G.).

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
php12018-sup-0001-FigureS1.pdfapplication/PDF245KFigure S1. In vitro phototoxicity experiments. (a) Phototoxicity experiments with A-431/G4 cells showing that PPa is highly phototoxic in vitro, whereas Ac-sPPp is relatively poorly phototoxic in vitro. Note that increasing the incubation period from 16 to 40 h did not increase the phototoxicity of Ac-sPPp. (b) Phototoxicity experiments showing that at a moderate PDT dose of 150 nm PPa equivalents and 10 J cm−2 of 670 nm light, anti-EGFR Ac-sPPp-cetuximab PIC is highly phototoxic toward EGFR-overexpressing A-431/G4 cells, while the control anti-HER2 Ac-sPPp-trastuzumab PIC exhibits no phototoxicity. In addition, competition of Ac-sPPp-cetuximab PIC with a four-fold greater concentration of native cetuximab largely inhibits all cell killing, demonstrating that Ac-sPPp-cetuximab PIC phototoxicity is mediated through specific binding of the EGFR under the specified moderate PDT treatment conditions. (c) Phototoxicity experiments showing that anti-EGFR Ac-sPPp-cetuximab PIC is only mildly to moderately phototoxic to non-target low EGFR-expressing SK-BR-3 cells, whereas PPa is highly phototoxic. Ac-sPPp-cetuximab phototoxicity towards SK-BR-3 cells is not fully inhibited by competition with native cetuximab, indicating that these cells also take up PIC by nonspecific processes such as adsorptive pinocytosis and/or phagocytosis. (d) Phototoxicity experiments with A-431/G4 cells showing that PIC targeting becomes nonspecific at relatively high PDT doses. For these experiments, cells were incubated with Ac-sPPp or anti-erbB Ac-sPPp PIC for 16 h. The light dose was 20 J cm−2 unless indicated otherwise. Note that at 150 nm PPa equivalents and 20 J cm−2 light dose, PIC targeting still exhibits a high degree of specificity as Ac-sPPp-cetuximab PIC phototoxicity is markedly inhibited by competition with the corresponding native cetuximab but not by competition with negative control trastuzumab. However, at relatively high PDT doses (≥300 nm PPa equivalents and ≥20 J cm−2 light dose), targeting specificity is lost, and PIC phototoxicity is not appreciably inhibited by competing native antibody. Error bars indicate standard deviations and are shown for data that was collected at least in triplicate.
php12018-sup-0002-FigureS1.pdfapplication/PDF4639KFigure S2. Fluorescence time-course imaging with Ac-sPPp-cetuximab PIC. A mouse bearing an A-431 tumor xenograft implanted subcutaneously in the left pectoral region was injected via the tail vein with Ac-sPPp-cetuximab PIC in PBS. Imaging experimental conditions were the same as those described in Fig. 2 where corresponding time-course imaging data for Ac-sPPp and the parent PS, PPa, are shown for comparison.
php12018-sup-0003-FigureS3.pdfapplication/PDF4397KFigure S3. Repeat PDT treatment of A-431 tumor-bearing mice using Ac-sPPp or Ac-sPPp-cetuximab PIC with light doses administered 40 h post-PS injection. Treatment conditions were similar to those described in Fig. 3 except the PS dose given for each individual PDT session was 5 nmol PPa equivalents per g body weight (instead of 3 nmol), and mice were PDT irradiated 40 h post-PS injection (instead of 16 h). (a) Tumor growth response for mice treated with repeat Ac-sPPp PDT (open triangles) or repeat Ac-sPPp-cetuximab PDT (filled triangles). Initial PDT was given day zero and then repeated 6 days later for Ac-sPPp PDT or 8 days later for Ac-sPPp-cetuximab PDT (arrows indicate the timing of the second treatment). Number of mice for each treatment regimen is indicated by n. For comparison, tumor growth curves for a group of untreated control mice (no PS and no light, gray open circles; = 10) are shown along with an average exponential fit of these data (thick line). (b) Representative photographs depicting tumor and surrounding normal tissue response of a mouse over the course of treatment with repeat Ac-sPPp-cetuximab PDT. One of the tumor growth response curves shown in (a) corresponds to this mouse. (c) Fluorescence imaging before and after the first round of Ac-sPPp-cetuximab PDT for the same mouse shown in (b). Photobleaching post-PDT is most prominent at the irradiated tumor site but is also evident elsewhere throughout the animal.

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