Photodynamic Therapy
In Vivo Studies of Nanostructure-Based Photosensitizers for Photodynamic Cancer Therapy Siew Hui Voon, Lik Voon Kiew, Hong Boon Lee, Siang Hui Lim, Mohamed Ibrahim Noordin, Anyanee Kamkaew, Kevin Burgess, and Lip Yong Chung*
From the Contents 1. Introduction .............................................. 2
Animal models, particularly rodents, are major
2. Roles for Nanoparticle Delivery Systems in PDT ........................................................ 2
translational models for evaluating novel anticancer therapeutics. In this review, different types of nanostructure-based photosensitizers that have advanced into the in vivo evaluation stage for the photodynamic therapy (PDT) of cancer are described. This article focuses on the in vivo efficacies of the nanostructures as delivery agents and as energy transducers for photosensitizers in animal models. These materials are useful in overcoming solubility issues, lack of tumor specificity, and access to tumors deep in healthy tissue. At the end of this article, the opportunities made possible by these multiplexed nanostructure-based systems are summarized, as well as the considerable challenges associated with obtaining regulatory approval for such materials. The following questions are also addressed: (1) Is there a pressing demand for more nanoparticle materials? (2) What is the prognosis for regulatory approval of nanoparticles to be used in the clinic?
3. Liposomes................................................. 3 4. Micelles ..................................................... 4 5. Polymer-based Nanoparticles .................... 9 6. Lipoprotein Nanoparticles ....................... 11 7. Inorganic Nanoparticles ........................... 11 8. Hybrid Nanoparticles ............................... 13 9. Combination Therapy (PDT and Chemotherapy) ........................................ 14 10. Upconverting Nanoparticles (Energy Transducers) ............................................ 15 11. Nanoparticles with Photosensitizing Properties................................................ 15 12. Conclusion ............................................. 17
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1. Introduction Photodynamic therapy (PDT) emerged as a treatment modality for neoplastic and non-malignant lesions after it was reported that a combination of red light and porfimer sodium (hematoporphyrin derivatives) could eradicate mouse mammary tumor growth.[1] Several clinical trials were then initiated, and PDT was shown to be successful in treating patients with bladder cancer and skin tumors.[2] Subsequent clinical studies involved other types of cancers, including those of the breast, colon, prostate, squamous cells, basal cells, endometrium, malignant melanoma, mycosis fungoides, chondrosarcoma and angiosarcoma.[3] PDT applications involve a photosensitizer, light and molecular oxygen. Upon light irradiation, the photosensitizer is activated and generates highly reactive singlet oxygen from molecular oxygen.[3a,4] Due to its short half-life of about 3 µs, damage caused by singlet oxygen is highly localized.[5] In cancer treatment, PDT can destroy tumor cells directly, damage vasculature surrounding tumor cells and activates immunological responses against tumors.[6] PDT using porfimer sodium (Photofrin; Axcan Pharma Inc., Mont-Saint-Hilaire, Canada) was approved for the treatment of bladder cancer in Canada in 1993. Since that time, other anticancer photosensitizing drugs (photosensitizers) have obtained clinical approval, including Foscan (temoporfin, meta-tetrahydroxyphenylchlorin; Biolitec AG), Visudyne (verteporfin, benzoporphyrin derivative monoacid ring A; Novartis Pharmaceuticals), Levulan (5-aminolevulinic acid; DUSA Pharmaceuticals, Inc.), and most recently, Metvix (methyl aminolevulinate; PhotoCure ASA.). PDT has remained at best a fringe cancer treatment option as it is limited by inaccessibility of deep seated tumors, skin photosensitivity for prolonged periods following treatment, high initial setup cost and lack of standard protocols established by randomized trials.[7] For instance, porfimer sodium causes lasting skin photosensitivity for up to 4–12 weeks at its therapeutic dose.[8] Many of the photosensitizers currently used cannot be excited by light of wavelengths >700 nm; this limits their use because tissue penetration of light into deeper tissues is only possible for long wavelength excitation sources. Although light-tissue interaction is not fully understood, wavelengths of less than 600 nm penetrate tissue to about 0.5 cm while slightly longer wavelengths up can double tissue penetration to 1 cm.[9] In the case of porfimer sodium, the relatively low maximum absorption wavelength of 630 nm has the challenge of poor tissue penetration of light while the very low extinction coefficient (1.170 M−1cm−1)[4] at this Q-band wavelength would increase the dose of drug and/or light required to give adequate phototherapeutic response: neither of these are the characteristics of an ideal clinical photosensitizer.[10] Moreover, most photosensitizer molecules are hydrophobic and aggregate easily in aqueous media, leading to reduced singlet oxygen generation[11] and difficulties with intravenous delivery strategies.[12] The other limitations may remain in the initial period until the availability of cheaper lasers and optic fiber equipment to reduce the high initial setup cost and standardization of treatment protocols for a greater range of target organs.[7]
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Ideal photosensitizers would be activated at >700 nm and have better tumor specificities, which would reduce the generalized photosensitivities.[13] Newer photosensitizers in clinical trials include tin ethyl etiopurpurin (SnET2), monoL-aspartyl chlorin e6 (Npe6), benzoporphyrin derivative (BPD), and lutetium texaphyrin (Lu-Tex); these have absorption bands at 660, 664, 690 and 732 nm, respectively, and provoke only mild and transient skin photosensitivity. However, clinical studies using these photosensitizers are restricted to the treatment of cutaneous cancer,[14] early squamous cell carcinoma of the lung,[15] and recurrent prostate cancer;[16] therefore, full understanding of the relative merits of these sensitizers and approval for general clinical use are still pending. Figure 1 presents chemical structures for many of the photosensitizers discussed in this review.
2. Roles for Nanoparticle Delivery Systems in PDT The effectiveness of PDT is not only largely determined by the efficiency of singlet oxygen generation[17] upon light activation, but also the efficiency and selectivity to which therapeutic concentrations of the photosensitizer is delivered to the targeted tumor site with minimal uptake by non-targeted normal cells.[18] Incorporating PDT agents into nanoparticles can alter these properties to ultimately improve clinical outcome. Covalent conjugation or physical inclusion of photosensitizers to nanoparticle carrier systems can surround hydrophobic photosensitizers with a hydrophilic environment, improving their solubilities and preventing aggregation in blood. This process may also reduce the non-specific phototoxicity to the skin and eyes, enhance the antitumor efficacy,[19] and increase passive tumor accumulation via an enhanced permeability and retention (EPR) effect.[7] This will elevate levels of nanostructure-based photosensitizers either in proximity to the tumor tissue or inside the cells
S. H. Voon, Dr. L. V. Kiew Department of Pharmacology Faculty of Medicine University of Malaya 50603, Kuala Lumpur, Malaysia Dr. H. B. Lee, S. H. Lim, Dr. M. I. Noordin, Prof. L. Y. Chung Department of Pharmacy Faculty of Medicine University of Malaya 50603, Kuala Lumpur, Malaysia E-mail: [email protected]; [email protected] Dr. H. B. Lee, S. H. Lim Cancer Research Initiatives Foundation (CARIF) Subang Jaya Medical Center 47500, Subang Jaya, Selangor, Malaysia A. Kamkaew, Prof. K. Burgess Department of Chemistry Texas A&M University Box 30012, College Station, Texas 77842, USA DOI: 10.1002/smll.201401416
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In Vivo Studies of Photosensitizers for Cancer Therapy
after internalization. Majority of cellular internalization of nanoparticles occur through pinocytosis known as clathrinmediated endocytosis (CME).[20] The attachment of cancertargeting moieties such as folic acid,[21] F3 peptide[22] or RGD peptide[23] to the nano-drug carriers can further enhance the selectivity for tumors. Chemical modification of the structures of the nano-drug carriers may also confer alternate means for energy transfer to photosensitizers, which enables the use of deep tissue-penetrating light for the activation of the photosensitizers to treat deeply seated tumors.[24] The concept of subcellular targeted nanoparticles is being developed and only a limited number of strategies has been identified for endoplasmic reticulum, mitochondria and nucleus targeting. Further investigations are necessary to evaluate its impact and relevance for future clinical applications. The design of subcellular targeted nanoparticles with non-immunogenic stealth character, with the ability to overcome biological barriers and target site specificity, remains necessary.[20a] This review describes examples of how photosensitizers in nanostructures have advanced into the in vivo evaluation stage, focusing on the in vivo efficacies of the nanostructures as delivery agents and as energy transducers for photosensitizers in animal models (Table 1). We also summarize the in vivo characteristics of nanostructures in terms of biodegradability, clearance and side effect as well as the regulatory approval status for clinical use (Table 2). This article complements previous reviews on the design, synthesis, physicochemical properties, and in vitro therapeutic efficiencies of photosensitizers.[25]
Siew Hui Voon obtained her BSc in Biomedical Science from National University of Malaysia in 2009. She received a MyPhD scholarship from Ministry of Education Malaysia in 2012 to pursue her PhD under the guidance of Prof. Lip Yong Chung at the University of Malaya. Her current research of interest is in the field of nanomaterials for drug delivery in photodynamic cancer therapy.
Lik Voon Kiew obtained his doctorate in biopolymer technology and therapeutics from the University of Malaya, Malaysia. He is currently senior lecturer at the Pharmacology department, Faculty of Medicine of the University of Malaya, Malaysia. His current research interests include the development and in vitro/in vivo evaluation of targetable anticancer polymer therapeutics, renal targeting drug carriers, anticancer photodynamic therapeutics, and bioactive compounds for chronic disease treatment.
Lip Yong Chung received his doctorate in pharmacy from the University of Cardiff and joined Cardiff University as a research associate focusing on bioactive polymers and cell regeneration, and is currently at the Faculty of Medicine of the University of Malaya, Ma-
3. Liposomes
laysia as a Professor in Pharmaceutical Sciences. His current research interests include
Liposomes are spherical, closed membranes composed of concentric phospholipid bilayers with an aqueous inner compartment.[26] They can accommodate lipophilic photosensitizers by incorporating them into the lipid bilayer, which renders them hydrosoluble and prevents aggregation in an aqueous environment (Figure 2).[27] These structures naturally tend to accumulate in tumors, which enhances the ability of incorporated photosensitizers to target the tumor tissue.[28] Several studies have demonstrated a higher accumulation of photosensitizers and better PDT efficacy in tumor tissues for photosensitizer-containing liposomes compared to free photosensitizers.[29] Thus, the incorporation of Photofrin into liposomes resulted in 6 and 2.4-fold increases in in vivo tumoral accumulation for 9L gliosarcoma and U87 glioma nude mice models, respectively, compared to the free Photofrin control in a dextrose formulation. Tissue necrosis was significantly increased in both tumor types after PDT compared to the controls.[30] Enhanced tumor accumulation of hypocrellin A was also observed when a liposome-hypocrellin A nanoconstruct was tested in an S-180 sarcoma Kunming mouse model, with maximum accumulation in tumor at 12 h (0.48 µg/g) compared to the group treated with hypocrellin A-DMSO (0.14 µg/g).[31] This observation is consistent with the higher tumor regression at day 7 after PDT (relative regression percent of tumor, small 2014, DOI: 10.1002/smll.201401416
the discovery of anticancer and CNS active compounds, and development of nanodrug conjugates for therapy.
RRP = 87%) and with the PDT efficacy exhibited by the liposome-hypocrellin A-treated group relative to the hypocrellin A-DMSO treatment group (RRP = 14%). Metatetra(hydroxyphenyl) chlorin formulated in unilamellar dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol liposomal nanoconstructs has been applied to female Foxn1nu/nu mice implanted with EMT-6 mammary tumor cells. In the four month follow-up period, 79% of the primary tumors were cured and showed no sign of recurrence.[32] Visudyne, a commercially available liposomal formulation of benzoporphyrin derivative monoacid ring A (BPDMA) indicated for age-related macular degeneration[33] when applied to Meth-A sarcoma-bearing mice using antiangiogenic scheduling (short drug-light interval of 15 min) showed greater tumor suppression compared to conventional scheduling (longer drug-light interval of 3 h). In this, 40 and 60% of the tumor of Meth-A sarcoma-bearing mice were cured
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Figure 1. Some photosensitizers featured in this review.
at 0.25 and 0.5 mg/kg BPD-MA, respectively. These suggest Visudyne is effective in cancer PDT using antiangiogenic scheduling.[34] These liposomes of 70–100 nm led to an increase in passive accumulation in the tumors via the enhanced permeability and retention effect. However, the main drawback of conventional liposomes is that they are easily taken up by cells of the reticuloendothelial system after systemic administration. This results in their rapid removal from the blood into the liver and spleen, which in turn reduces the accumulation in the tumor tissue.[35] Some liposome modifications have been reported to counteract these delivery problems. For instance, polyethylene glycol has been added to liposomal surfaces to create more stable “sheathed” forms. Such PEGylation may protect liposomes from being recognized by opsonins and taken up by the reticuloendothelial system.[36] However, the increased stability provided by this type of
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modification of the liposomes may also decrease the liposome-cell interactions and reduce the transfer of the photosensitizer payload into tumor cells.[37]
4. Micelles Micelles are aggregates of surfactant molecules in liquid-colloid dispersions in which the hydrophilic head regions are in contact with surrounding solvent and the hydrophobic tails are in the center. The particle size range is normally within 5–100 nm. They are widely used to carry hydrophobic drugs, physically entrapped in or covalently bound to the hydrophobic core and delivered to tumors via passive or active targeting strategies.[38] Micelles can be classified according to the nature of the amphiphilic core: polymeric or lipid micelles. Polymeric micelles are formed from block copolymers,
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Table 1. In Vivo Studies Reported on Nanostructure-Based Photosensitizer Formulations. Nanostructure
Photosensitizer
Animal model
Photofrin (porfimer sodium); hyprocrellin A; metatetra(hydroxyphenyl) chlorin (mTHPC); Verteporfin
Athymic (nude) rats (strain Cr: NIH-rna) implanted with U87 glioma cells through craniectomy;[30a] male Kunming mice transplanted with S-180 sarcoma;[31] female Foxn1nu/nu mice injected subcutaneously with EMT6 cells;[32] male BALB/c mice injected subcutaneously with Meth-A sarcoma cells[34]
Polymer Micelles
Aluminum chloride phthalocyanine (AlClPc); protophorphyrin IX (PpIX)
Mice with squamous cell carcinoma (SCC7) tumor;[40] Balb/c mice implanted with EMT-6 tumors[41]
PEG-Lipid Micelles
Verteporfin; mesotetraphenylporphine (TPP)
Female C57BL/6 mice injected subcutaneously with LLC cells;[43a[ male DBA/2 mice implanted with rhabdomyosarcoma (M1) tumor cells[46]
Cremophor EL
Temocene; azabodipy
DBA/2 mice inoculated with the DBA/2 mastocytoma cell line P815;[53] Balb/c mice inoculated with the Balb/c colon adenocarcinoma cell line CT26.CL25 (ATCC, CRL-2639);[53] female Balb/c nu/nu mice injected with MDA-MB-231-GFP cells[51]
Poly(lactic-co-glycolic acid) (PLGA)
Zinc phthalocyanine; verteporfin
Female albino mice injected with Ehrlich ascites carcinoma cells;[56] male DBA/2 mice implanted with rhabdomyosarcoma (M1) tumor[18]
Dendrimers
Aminolevulinic acid (ALA); phthalocyanine
Male BALB/c mice injected with the LM3 cell line;[60] female nude mice (BALB/c nu/nu) subcutaneously transplanted with subcutaneous A549 tumor cells[61]
Chitosan
Protophorphyrin IX (PpIX); chlorin e6
Athymic nude mice inoculated with SCC7 cells;[65] athymic nude mice injected with HT-29 human colon adenocarcinoma cells[67,70[
LIPOPROTEIN NANOPARTICLES
Bacteriochlorin e6 bisoleate (Bchl-BOA)
Female athymic nude mice inoculated with HepG2 cells[77]
Silica
Protophorphyrin IX (PpIX); methylene blue; zinc phthalocyanine
Male athymic Nude-Foxn1nu mice subcutaneously implanted with glioblastoma multiforme;[82] female athymic Swiss nude mice and female athymic Naval Medical Research Institute nude mice injected subcutaneously with HCT 116 and A549 cells, respectively;[82] male athymic BALB/c (Balb/C-nu) mice inoculated subcutaneously with Hela cells;[80] female BALB/c nude mice implanted with H22 cells;[84] female Swiss nude mice xenografted with HCT-116 cells[85]
Gold
Silicon phthalocyanine 4; porphyrinbrucine conjugates; Chlorin e6
NuNu mice subcutaneously injected with basaloid squamous cell carcinoma PE/CA-PJ34 cells;[90a] mice with MDA-MB-435 tumor[92]
Calcium Phosphosilicate
Indocyanine green (ICG)
Female C3H/HeJ mice injected with 32D-p210-GFP cells[94]
Photofrin (porfimer sodium)
Male Fischer 344 rats implanted with rat 9L glioma cells[22]
N-(2-Hydroxypropyl)methacrylamide (HPMA)
meso-chlorin e6 monoethylene diamine (Mce6)
Female nu/nu mice with OVCAR-3 solid tumor[102b]
Liposome
Doxorubicin (Chemotherapeutic agent); Verteporfin
Female BALB/c mice inoculated subcutaneously with murine colon carcinoma Colo 26;[103] Male Copenhagen rats implanted subcutaneously and orthotopically with R3327-MatLyLu Dunning prostate tumor cells[104]
UPCONVERSION NANOPARTICLES (ENERGY TRANSDUCER)
Chlorin e6; merocyanine 540 (MC540); zinc phthalocyanine (ZnPc)
Female Balb/c mice injected subcutaneously with 4T1 cells;[24b] C57BL/6 female mice implanted subcutaneously with B16-F0 cells[21]
Titanium Dioxide
None
Nude mice injected subcutaneously with T-24 cells;[108] female BALB/c-nude mice inoculated with U87 cells[110]
Fullerenes (C60)
None
Female CDF1 mice inoculated with Meth A fibrosarcoma cells;[118a] female CDF1 mice inoculated with Meth AR1fibrosarcoma cells;[118b] male BALB/c mice inoculated with CT26-Luc cells;[122] mice xenografted with human-melanoma (COLO679)[123]
LIPOSOMES
MICELLES
POLYMER-BASED NANOPARTICLES
INORGANIC NANOPARTICLES
HYBRID NANOPARTICLES Polyacrylamide COMBINATION THERAPY (PDT & CHEMOTHERAPY)
NANOPARICLES WITH PHOTOSENSITIZING PROPERTIES
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Table 2. In Vivo Characteristics of Nanostructures and Their Regulatory Approval Status for Clinical Use. Nanostructure
Biodegradability
Metabolism & Clearance
Side Effects
FDA Approval Status for PDT use
Regulatory Approval Status for other anticancer drugs
LIPOSOMES
Yes
Vesicle opsonization by serum protein and subsequent uptake by the reticuloendothelial system; Complementmediated phagocytosis by Kupffer and endothelial cells of the liver as well as other phagocytic cells of the reticuloendothelial system[128]
NA
Yes
Clinically approved: Doxil (Doxorubicin) [US, EU][129]
Clinically approved: Visudyne (Verteporfin) to treat age-related macular degeneration
DaunoXome (Daunorubicin citrate) [US][129]
Phase I & II clinical trials: CGP 55847 (liposomal zinc(II)phthalocyanine) for cancer treatment[125]
Depocyt (Cytarabine) [US, EU][130] Mepact (Mifamurtide) [EU][131] Marqibo (Vincristine sulfate) [On market][132]
MICELLES Polymer Micelles
No
Degradation of polymer micelles, resulting in the formation of block copolymer unimers, which can be removed via renal excretion if the polymer chains are designed with a lower molecular weight than the critical value for renal filtration less than ∼20–40 kDa.[133]
Slow extravazation; Risk of chronic liver toxicity due to prolonged circulation and slower metabolism of drug which may exhibit toxic side effects[133]
No
Clinically approved: Taxotere (Docetaxel) [EU, US][129a] Phase II clinical trials: SP1049C (Pluronic block-copolymer doxorubicin)[129a,134]
PEG-Lipid Micelles
NA
NA
NA
No
NA
Cremophor EL
Slow
May be largely degraded in the blood compartment by serum carboxylesterase-induced degradation, causing a gradual release of the ricinoleic acid residues attached to the triglyceride structure; Hepatobiliary elimination; Less than 0.1% of administered dose via urinary excretion[52]
Associated with severe anaphylactoid hypersensitivity reactions, hyperlipidaemia, abnormal lipoprotein patterns, aggregation of erythrocytes and peripheral neuropathy[52]
No
Clinically approved: Taxol (Paclitaxel) [US][129a]
Cremophor EL
Slow
May be largely degraded in the blood compartment by serum carboxylesterase-induced degradation, causing a gradual release of the ricinoleic acid residues attached to the triglyceride structure; Hepatobiliary elimination; Less than 0.1% of administered dose via urinary excretion[52]
Associated with severe anaphylactoid hypersensitivity reactions, hyperlipidaemia, abnormal lipoprotein patterns, aggregation of erythrocytes and peripheral neuropathy[52]
No
Clinically approved: Taxol (Paclitaxel) [US][129a]
Undergo hydrolysis and biodegrades into lactic and glycolic acids. Lactic acid enter the tricarboxylic acid cycle and is metabolized and subsequently eliminated from the body as carbon dioxide and water. Glycolic acid is either excreted unchanged in the kidney or enters the tricarboxylic acid cycle and is eventually eliminated as carbon dioxide and water[55a]
NA
Approved by the US FDA for the use of drug delivery[135]
Phase II Clinical Trials: WST09 (TOOKAD) in Cremophor EL formulation[126a] Phase I Clinical Trias: Silicon Phthalocyanine Pc 4 in Cremophor EL formulation[126b]
POLYMER-BASED NANOPARTICLES Poly(lactic-coglycolic acid) (PLGA)
Yes
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NA
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Table 2. Continued Nanostructure
Biodegradability
Metabolism & Clearance
Side Effects
FDA Approval Status for PDT use
Regulatory Approval Status for other anticancer drugs
Dendrimers
NA
Renal clearance for dendrimers with diameter 3–10 nm[128]
NA
No
Phase III clinical trials: SH L 643A (Gadolinium) for diagnostic imaging[136]
Chitosan
Yes
Mainly degraded by lysozyme through the hydrolysis of the acetylated residues[137]
NA
No
NA
LIPOPROTEIN NANOPARTICLES
Yes
Catabolized by the endothelium-associated lipoprotein lipase, thereby generating free fatty acids, which are taken up by the liver, muscle, and adipose tissues[138]
NA
No
NA
Silica
Slow
Gradual biodegradation results in the formation of water-soluble salts of silicic acid, which are excreted by the kidney; Hepatobiliary excretion[139]
Caused granuloma formation in the organs of the reticulo-endothelial system, such as liver and spleen[139,140]
No
NA
Gold
No
Renal clearance for particles with diameter < 10 nm[128]
NA
No
Phase I clinical trials: AuroShell (gold nanoparticle) for laser therapy[141]
Calcium Phosphosilicate
No
Hepatobiliary clearance with minimal acute renal involvement[93a]
NA
No
NA
Yes
Phagocytosis by Kupffer cells followed by hepatobiliary and renal clearance[97]
NA
No
NA
N-(2-Hydroxypropyl) methacrylamide (HPMA)
No
Blood clearance by spleen macrophage and eliminated by glomerular filtration in the kidney[137]
NA
No
Phase II clinical trials: ProLindac (HPMA copolymer–DACH platinate)[142]
UPCONVERSION NANOPARTICLES (ENERGY TRANSDUCER)
No
Hepatobiliary clearance[24b]
NA
No
NA
Titanium Dioxide
No
94% and 2% of administered TiO2 was found in the liver and spleen and did not decrease up to 30 days after administration[143]
Can induce pathological lesions of the liver, spleen, kidneys, and brain[144]
No
NA
Fullerenes (C60)
No
Increased hepatobiliary accumulation and excrete via urinary tract[114,128]
NA
No
NA
INORGANIC NANOPARTICLES
HYBRID NANOPARTICLES Polyacrylamide
COMBINATION THERAPY (PDT & CHEMOTHERAPY)
NANOPARICLES WITH PHOTOSENSITIZING PROPERTIES
NA = not available
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Figure 2. Liposome. Size: 70–100 nm.
whereas lipid micelles are prepared from water-soluble polymers conjugated to lipids.
4.1. Polymer Micelles Polymer micelles are formed in an aqueous solution from amphiphilic block or graft copolymers consisting of hydrophilic and hydrophobic monomeric units, which form their corona and core, respectively (Figure 3a). These self-assembling structures have the ability to solubilize drugs with poor water solubility,[39] and uptake by the reticuloendothelial system is substantially reduced by the hydrophilic palisades of tethered polymer chains surrounding the drug-loaded core.[39d] The uptake of pH-responsive methoxy polyethylene glycol-poly(β-amino ester) block copolymer micelles (pHPMs) encapsulating protophorphyrin IX (PpIX) by the reticuloendothelial system has been shown to be reduced.[40] In SCC7 tumor-bearing mice, biodistribution evaluation measured by photon counts showed that the PpIX level in tumors for the PpIX-pH-PMs treated group were 10× higher than that of free PpIX treated group at 48 h post-injection. Ex vivo imaging of the excised organs (liver, lung, spleen, kidney, heart) and tumors showed the strongest fluorescence intensity in the tumors, while PpIX uptake in other normal organs was not significant except in liver and kidney, organs where PpIX is rapidly metabolized. In contrast, when free PpIX was delivered, strong fluorescent signals were observed mainly in the liver, whereas the tumor only presented a very weak fluorescent signal that could not be clearly distinguished from the body. In this study, complete ablation of tumors occurred in mice treated with PpIX-pH-PMs, whereas tumor growth continued in free PpIX-treated group. Histological examinations revealed that most of the tumor cells in the mice treated with PpIX-pH-PMs were severely damaged or destroyed at day 10 after treatment, while incomplete tumor cell death was observed in the mice treated with free PpIX. N-iso-Propylacrylamide (NIPAM) copolymer micelles have been co-polymerized with a pH-sensitive moiety, methacrylic acid and N-vinyl-2-pyrrolidone units to impart
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Figure 3. (a) Polymer micelle. Size: 30–122 nm; (b) PEG-lipid micelles. Size 13–30 nm; (c) Cremophor EL. Size: 30 nm.
tumor-targeting properties and stealth properties, respectively.[41] The methacrylic acid is believed to increase tumoral localization, while the N-vinyl-2-pyrrolidone provides a nonionic hydrophilic shield to reduce uptake by the mononuclear phagocytes, thus increasing the time the micelles remain circulating in the blood. When loaded with aluminum chloride phthalocyanine (AlClPc), these AlClPc-polymer micelle formulations did not show increased tumoral uptake of AlClPc over the Cremophor® EL control formulation (1.8–2% vs. 2.4% AlClPc of injected dose/g, respectively). Both formulations showed similar PDT efficacy with no tumor recurrence in 80% of mice bearing intradermal EMT-6 tumors. However, the low toxicity of these modified NIPAM polymeric micelles and their strong affinity for AlClPc makes them a good alternative to Cremophor EL for the administration of poorly water-soluble phthalocyanines for PDT.
4.2. Polyethylene Glycol–Lipid Micelles Polyethylene glycol-lipid micelles consist of polyethylene glycol conjugated to a relatively short but hydrophobic diacyl phospholipid moiety such as phosphatidyl ethanolamine (Figure 3b).[42] In this construct, the phosphatidyl ethanolamine binds to the hydrophobic photosensitizers[43] and the
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In Vivo Studies of Photosensitizers for Cancer Therapy
polyethylene glycol prevents the rapid uptake of the micelle nanoparticles by the reticuloendothelial system.[44] Roby et al.[43a] modified the polyethylene glycol-lipid micelles by attaching antibodies to the micellar surface as tumor-targeting ligands for meso-tetraphenylporphine (TPP) delivery. Specifically, they used the monoclonal antibody 2C5 (MAb 2C5) to target surface-bound nucleosomes, which are released from apoptotically dying cancer cells. Two-tothree-fold improved accumulation of TPP in tumors was achieved compared to the non-targeted polyethylene glycolphosphatidyl ethanolamine micelles in female C57BL/6 mice injected subcutaneously with Lewis lung carcinoma cells. The antibody-targeted micelles resulted in complete inhibition of tumor growth until day 35 after PDT treatment, while those lacking the antibody only reduced tumor growth by 50%. Treatment with free TPP only caused slight tumor growth suppression compared to the untreated control. Histological examination of the tumors showed significantly higher tumor cell death in the mice treated with TPP-loaded MAb 2C5-polyethylene glycol-phosphatidyl ethanolamine-immunomicelles. Formulation of benzoporphyrin photosensitizers in methoxypoly(ethylene glycol)-distearoylphosphatidylethanolamine (mPEG-DSPE) micelles also increased their solubility.[45] An intravenous dose of 1.4 µmol/kg of an mPEG2000-DSPE micellar formulation of an A-ring benzoporphyrin derivative at a 6:1 lipid/drug ratio caused tumor regression in male DBA/2 mice implanted with rhadbomyosarcoma (M1) tumor cells. The mice remained free of tumors up to 20 days after PDT, while there was no observable tumor reduction in the control mice.[46]
4.3. Cremophor EL Cremophor EL is a self-assembling micelle containing glycerol polyethylene glycol ricinoleate used to solubilize hydrophobic photosensitizing agents and promote their distribution into plasma lipoproteins (Figure 3c).[47] It has been used in combination with ethanol to solubilize lipophilic photosensitizers, such as TOOKAD,[48] phthalocyanine,[49] purpurin[50] and azabodipy-based photosensitizers.[51] The in vivo use of Cremophor EL has been associated with anaphylactic hypersensitivity reaction and neurotoxicity,[52] but it is widely used in early preclinical studies because it conveniently and efficiently emulsifies lipophilic entities. Temocene incorporated in Cremophor EL micelles showed a better in vivo response than free temocene. Total regression of the principal tumors and remission of tumors in Balb/c mice subcutaneously inoculated with CT26.CL25 tumor cells and DBA/2 mice inoculated with mastocytoma cells P815 for 40 and 60 days, respectively, were observed.[53] The best PDT response was obtained if the photoactivation was carried out after a short drug-light interval of 15 min after injection of the temocene-loaded micelles. Tumor regression was due to tumor-associated vascular damage leading to tumor hypoxia. The kinetics of the drug accumulation suggests that the small Cremophor EL micellar size (30 nm) confers fast tumoral accumulation, and the micelles are small 2014, DOI: 10.1002/smll.201401416
then rapidly cleared by the reticuloendothelial system. This temocene-loaded Cremophor EL micellar formulation significantly improves the PDT effect of temocene and is particularly suited for vascular-targeted treatments. Cremophor EL micelles were also used for the delivery of a lead BF2-azadipyrromethene molecule, ADMP06, targeting the mammary tumor vasculature[51] and were evaluated in nude mice inoculated subcutaneously with MDA-MB-231-GFP cells. After PDT treatment, tumor ablation was demonstrated for 71% of the treated nude mice with no recurrence for 6 months.
5. Polymer-based Nanoparticles Some polymer-based nanoparticles can have good aqueous dispersions, high drug-loading capacities, and extended release properties. Modifications are possible to alter their particle size, surface characteristics, and ability to achieve active or passive targeting.[54] Several categories of polymeric nanoparticles have been studied as carriers for PDT agents, including synthetic polymers e.g., poly(lactide-co-glycolide) copolymers (PLGA) and dendrimers, and natural polymers such as chitosan.
5.1. Poly(lactic-co-Glycolic Acid) (PLGA) PLGAs are copolymers of lactic and glycolic acids. These structures can have excellent biocompatibility, biodegradability and mechanical strength. They have been formulated into various delivery systems for carrying a variety of active agents such as vaccines, peptides, proteins, and macromolecules (Figure 4a). Some of these agents are now approved by the US Food and Drug Administration for use in drug delivery.[55] Fadel et al.[56] formulated PLGA nanoparticles to enhance tissue uptake, permeation, and targeting of zinc (II) phthalocyanine for photodynamic therapy. Female albino mice implanted with Ehrlich ascites carcinoma cells and treated with nanoparticle formulated-zinc (II) phthalocyanine survived significantly longer than those treated with free drug (mean = 60 and 25 days, respectively), while the mean survival time of an untreated control group was 15 days. Two weeks after the treatment, the mean tumor volumes for the nanoparticle formulated-zinc (II) phthalocyanine group were found to be 1.5 times smaller than those in animals treated with the free drug, and the animals in the former group exhibited a longer tumor growth delay of 39 days. PLGA nanoparticles have also been used to carry Verteporfin. Free Verteporfin causes extensive skin photosensitivity,[57] but male DBA/2 mice treated with Verteporfin loaded in PLGA nanoparticles showed only a very mild and short period of skin photosensitivity as measured by erythema/eschar formation and edema observations 24 h after PDT in a rhabdomyosarcoma (M1) tumor model.[18] Total tumor eradication with no tumor regrowth was observed up to 14 days after PDT and on day 20, only 34% of the mice showed tumor regrowth.
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of the dendrimer-conjugate for optimum cellular uptake. Many dendrimers also have good tissue biodistribution properties.[59] Casas et al.[60] used ester coupling reactions to synthesize a dendrimer bearing eighteen aminolevulinic acid residues (18m-ALA). This agent induced sustained porphyrin production that peaked at 24 h in male Balb/c mice in contrast to the response to free aminolevulinic acid, which peaked sharply at 3 to 4 h. The tumoral accumulation of 18m-ALA was 7.5 times higher for the 18m-ALA dendrimer than for free aminolevulinic acid at the same concentration. However, the in vivo efficacy was not determined. Use of polyionic micelles has been studied to explore the hypothesis that they could reduce π–π interactions of photosensitizers that tend to lead to aggregation.[61] Thus dendrimeric phthalocyanine complexed with poly-L-lysinepolyethylene glycol polymeric micelles (DPc/m) were evaluated in female Balb/cnu/nu mice subcutaneously implanted in the back with human lung adenocarcinoma A549 cells.[61] A significantly reduced tumor growth rate was observed for the mice treated with DPc/m compared to the dendrimeric phthalocyanine-treated and control (without drug treatment) groups. Moreover, the PDT effect of DPc/m was significantly greater than Photofrin alone, even though the injected dose of DPc/m, based on the photosensitizing units, was 7.3-fold lower than Photofrin. DPc/m did not induce phototoxicity after PDT treatment, whereas severe skin and liver damage was observed in Photofrin-treated mice.
5.3. Chitosan Nanoparticles
Figure 4. (a) Poly(lactic-co-glycolic acid) (PLGA) nanoparticle. Size: 117–450 nm; (b) Dendrimer. Size: 50 nm; (c) Chitosan nanoparticle. Size: 260–350 nm.
5.2. Dendrimers Dendrimers are regularly branched, three-dimensional structures composed of a central core molecule and branching units that can be conjugated to drug molecules (Figure 4b).[58] Dendritic branching terminals may be functionalized with solubilizing groups, targeting groups or other moieties. Dendrimers have the advantages of high drug payload and the ability to control and modify the size and lipophilicity
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Synthetic polymers are easily modified to achieve desirable properties, but natural polymers are of interest because they are biodegradable and readily available. Chitosan, a product of partial deacetylation of the natural polysaccharide chitin, is the second most abundant biopolymer after cellulose. This natural polysaccharide has been widely used in various biomedical and pharmaceutical applications,[62] due to its wide availability, bioavailability, low immunogenicity and suitability for chemical modifications (Figure 4c).[63] Glycol chitosan, a chitosan derivative with ethylene glycol groups added to its backbone to enhance its water solubility,[64] was used by Lee et al.[65] to physically encapsulate protophorphyrin IX in nanoparticles. In vivo tumor uptake of the protophorphyrin IX-loaded nanoparticles was 2.3× higher than free protophorphyrin IX in squamous cell carcinoma (SCC7)bearing athymic nude mice. Moreover, the total fluorescent photon counts per organ for the tumor tissues were 1.49 to 3.8fold higher than in other organs, implying favorable biodistribution properties. On day 14 after treatment, measurements of the tumor volumes showed a 44% reduction in the protophorphyrin IX-loaded nanoparticle-treated group, where the same regimen of free protophorphyrin IX did not produce substantial tumor volume reduction. Furthermore, histological examination showed that most of the tumor cells were severely damaged or destroyed in the mice treated with the protophorphyrin IX-loaded nanoparticles, whereas incomplete tumor death was observed in the mice treated with free protophorphyrin IX.
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In Vivo Studies of Photosensitizers for Cancer Therapy
Nanoparticles in which the cargo is non-covalently incorporated can exhibit burst release profiles; such sudden liberation of cargo can reduce tumor targeting and increase damage to normal tissues.[66] To circumvent this, Lee et al.[67] used chemical conjugation rather than physical loading to prepare protophorphyrin IX-conjugated glycol chitosan nanoparticles. These particles exhibited desirable “safety catch” photodynamic properties because the highly dense protophorphyrin IX cores remain in a quenched ‘off’ state until they are taken up into cells; in their off-state they may not generate sufficient singlet oxygen to produce phototoxicity.[68] After cellular uptake, the dense protophorphyrin IX cores disintegrate in the intracellular environment,[69] permitting the protophorphyrin IX to revert to a photocytotoxic ‘on’ state. Thus, in mice bearing the HT-29 human colon adenocarcinoma, the photon counts in the tumor tissues of mice treated with the protophorphyrin IX-conjugated glycol chitosan nanoparticles were 1.4 or 2.1-fold higher than in the tumors of mice treated with protophorphyrin IX physically loaded into glycol chitosan nanoparticles and free protophorphyrin IX, respectively. The tumor sizes were at least 5.2-fold smaller in the mice treated with the protophorphyrin IX-conjugated glycol chitosan nanoparticles than in those treated with free protophorphyrin IX. Furthermore, histological examination revealed that little apoptosis and necrosis was observed in the tumors of mice treated with free protophorphyrin IX, in contrast to the large amount of cell death in the tumor tissues of those treated with the protophorphyrin IX-conjugated glycol chitosan nanoparticles. Lee et al.[70] supported his findings above by comparing the antitumor efficacy of chlorin e6 of chemically conjugated and physically loaded glycol-chitosan nanoparticles in HT-29 human colon adenocarcinoma tumor-bearing mice. Severe photo-induced tumor necrosis was induced in the mice treated with the conjugated chlorin e6-glycol-chitosan nanoparticles whereas the group treated with the physically loaded chlorin e6-glycol-chitosan nanoparticles failed to show noticeable phototoxicity in the tumor tissues. Final tumor volumes in the mice treated with the conjugated chlorin e6-glycol-chitosan-nanoparticles were approximately 160 mm3 at 20 days post-injection, which was significantly smaller than the size of the tumors (approximately 560 mm3) in the mice treated with the physically loaded chlorin e6-glycol-chitosan-nanoparticles. The improved antitumor efficacy of conjugated chlorin e6-glycol-chitosan nanoparticles in these experiments was attributed to more efficient accumulation of these particles in the tumors.[70] The fluorescence of the conjugated chlorin e6-glycolchitosan-nanoparticles in the tumor tissues persisted for two days after administration. In contrast, the physically loaded chlorin e6-glycol-chitosan nanoparticles showed no specific tumor accumulation: instead, an intense whole-body fluorescence at 3 h post-injection was followed by a rapid decrease in fluorescence. Furthermore, the total photon counts of the chemically conjugated chlorin e6-glycol-chitosan nanoparticles in tumor tissue were approximately 6 to 7-fold higher than that of the physically loaded chlorin e6-glycolchitosan nanoparticles. small 2014, DOI: 10.1002/smll.201401416
6. Lipoprotein Nanoparticles Lipoprotein nanoparticles are composed of naturally occurring apoproteins, phospholipids and cholesterol on surfaces that encapsulate cholesterol esters and triglycerides in the hydrophobic core.[71] They are potentially excellent cancer drug delivery systems because they can remain in circulation for an extended period while evading the reticuloendothelial system.[72] The hydrophobic core facilitates incorporation of poorly soluble photosensitizers, and these materials are amenable to various drug-linking strategies (Figure 5).[71] Low-density-lipoprotein (LDL) (60 days, and no tumors were present in two of the three rats after 6 months.
adriamycin to nude mice with human epithelial ovarian carcinoma. The HPMA copolymer-adriamycin conjugate with 2.2 mg/kg adriamycin equivalent plus HPMA copolymermeso-chlorin e6 at 1.5 and 8.7 mg/kg meso-chlorin e6 equivalent with light resulted in significant tumor regression relative to receiving either agent and/or with light alone. No shock syndrome was observed in the mice; this can result from massive agent-stimulated prostaglandin release, which causes platelet aggregation and damage and leads to obstruction of blood flow. This study suggests that an HPMA copolymer carrier can extend the safety margin of meso-chlorin e6 in vivo in PDT because free meso-chlorin e6 at between 2.5 and 10 mg/kg not only caused tumor regression but also caused shock syndrome. Synder et al. developed a combination therapy using PDT to enhance the delivery and efficacy of macromolecule-based cancer drug such as Doxil a pegylated liposome encapsulated doxorubicin with an average diameter of 100 nm.[103] Prior to the administration of Doxil, murine Colo 26 tumor-bearing mice was treated with the photosensitizer 2-[1-hexyloxyethyl]2-devinyl pyropheophorbide-a (HPPH) at 0.4 mol/kg and subjected to PDT at 48 J/cm2 and 14 mW/cm2. This increased the accumulation of doxorubicin in transplanted Colo 26 tumors significantly without concomitant enhancement of systemic or local toxicity. Eighty percent of tumor-bearing mice treated with the combination of PDT and Doxil at 5 mg/kg were cured compared to the untreated tumor-bearing control mice. On the other hand, a dose of just 5 mg/kg Doxil without PDT only showed modest tumor growth delay without complete remission. Furthermore, the combination of PDT and Doxil treatment also reduced skin toxicity in terms of erythema, broken vessels and light eschar formation. This phenomenon is supported by Bin Chen et al.’s findings in relation to subcutaneous and orthotopic MatLyLu rat prostate models, where tumor uptake of macromolecules such as Evans blue-albumin and high molecular weight FITC-labeled dextran was significantly increased following PDT treatment. This suggests that PDT increased vascular barrier dysfunction in the MatLyLu tumors. The observation on the adherence of blood cells to vessel wall shortly after PDT further supports the loss of endothelial integrity in the blood vessels.[104]
9. Combination Therapy (PDT and Chemotherapy) N-(2-Hydropropyl)methacrylamide (HPMA) is a water-soluble, biocompatible polymeric drug carrier that can be easily formulated to include targeting ligands[100] and has been shown to preferentially accumulate in tumors via the enhanced permeability and retention effect.[101] HPMA copolymer-bound drugs have been developed for the combination of PDT and chemotherapy (Figure 8).[102] Peterson et al.[102a] designed a combination of two HPMA nanoparticle systems to co-deliver meso-chlorin e6 and
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Figure 8. Combination therapy. Size: 25 nm. © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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In Vivo Studies of Photosensitizers for Cancer Therapy
10. Upconverting Nanoparticles (Energy Transducers) One way to overcome the limitations imposed by poor tissue penetration of shorter wavelength (
In Vivo Studies of Nanostructure-Based Photosensitizers for Photodynamic Cancer Therapy Siew Hui Voon, Lik Voon Kiew, Hong Boon Lee, Siang Hui Lim, Mohamed Ibrahim Noordin, Anyanee Kamkaew, Kevin Burgess, and Lip Yong Chung*
From the Contents 1. Introduction .............................................. 2
Animal models, particularly rodents, are major
2. Roles for Nanoparticle Delivery Systems in PDT ........................................................ 2
translational models for evaluating novel anticancer therapeutics. In this review, different types of nanostructure-based photosensitizers that have advanced into the in vivo evaluation stage for the photodynamic therapy (PDT) of cancer are described. This article focuses on the in vivo efficacies of the nanostructures as delivery agents and as energy transducers for photosensitizers in animal models. These materials are useful in overcoming solubility issues, lack of tumor specificity, and access to tumors deep in healthy tissue. At the end of this article, the opportunities made possible by these multiplexed nanostructure-based systems are summarized, as well as the considerable challenges associated with obtaining regulatory approval for such materials. The following questions are also addressed: (1) Is there a pressing demand for more nanoparticle materials? (2) What is the prognosis for regulatory approval of nanoparticles to be used in the clinic?
3. Liposomes................................................. 3 4. Micelles ..................................................... 4 5. Polymer-based Nanoparticles .................... 9 6. Lipoprotein Nanoparticles ....................... 11 7. Inorganic Nanoparticles ........................... 11 8. Hybrid Nanoparticles ............................... 13 9. Combination Therapy (PDT and Chemotherapy) ........................................ 14 10. Upconverting Nanoparticles (Energy Transducers) ............................................ 15 11. Nanoparticles with Photosensitizing Properties................................................ 15 12. Conclusion ............................................. 17
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1. Introduction Photodynamic therapy (PDT) emerged as a treatment modality for neoplastic and non-malignant lesions after it was reported that a combination of red light and porfimer sodium (hematoporphyrin derivatives) could eradicate mouse mammary tumor growth.[1] Several clinical trials were then initiated, and PDT was shown to be successful in treating patients with bladder cancer and skin tumors.[2] Subsequent clinical studies involved other types of cancers, including those of the breast, colon, prostate, squamous cells, basal cells, endometrium, malignant melanoma, mycosis fungoides, chondrosarcoma and angiosarcoma.[3] PDT applications involve a photosensitizer, light and molecular oxygen. Upon light irradiation, the photosensitizer is activated and generates highly reactive singlet oxygen from molecular oxygen.[3a,4] Due to its short half-life of about 3 µs, damage caused by singlet oxygen is highly localized.[5] In cancer treatment, PDT can destroy tumor cells directly, damage vasculature surrounding tumor cells and activates immunological responses against tumors.[6] PDT using porfimer sodium (Photofrin; Axcan Pharma Inc., Mont-Saint-Hilaire, Canada) was approved for the treatment of bladder cancer in Canada in 1993. Since that time, other anticancer photosensitizing drugs (photosensitizers) have obtained clinical approval, including Foscan (temoporfin, meta-tetrahydroxyphenylchlorin; Biolitec AG), Visudyne (verteporfin, benzoporphyrin derivative monoacid ring A; Novartis Pharmaceuticals), Levulan (5-aminolevulinic acid; DUSA Pharmaceuticals, Inc.), and most recently, Metvix (methyl aminolevulinate; PhotoCure ASA.). PDT has remained at best a fringe cancer treatment option as it is limited by inaccessibility of deep seated tumors, skin photosensitivity for prolonged periods following treatment, high initial setup cost and lack of standard protocols established by randomized trials.[7] For instance, porfimer sodium causes lasting skin photosensitivity for up to 4–12 weeks at its therapeutic dose.[8] Many of the photosensitizers currently used cannot be excited by light of wavelengths >700 nm; this limits their use because tissue penetration of light into deeper tissues is only possible for long wavelength excitation sources. Although light-tissue interaction is not fully understood, wavelengths of less than 600 nm penetrate tissue to about 0.5 cm while slightly longer wavelengths up can double tissue penetration to 1 cm.[9] In the case of porfimer sodium, the relatively low maximum absorption wavelength of 630 nm has the challenge of poor tissue penetration of light while the very low extinction coefficient (1.170 M−1cm−1)[4] at this Q-band wavelength would increase the dose of drug and/or light required to give adequate phototherapeutic response: neither of these are the characteristics of an ideal clinical photosensitizer.[10] Moreover, most photosensitizer molecules are hydrophobic and aggregate easily in aqueous media, leading to reduced singlet oxygen generation[11] and difficulties with intravenous delivery strategies.[12] The other limitations may remain in the initial period until the availability of cheaper lasers and optic fiber equipment to reduce the high initial setup cost and standardization of treatment protocols for a greater range of target organs.[7]
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Ideal photosensitizers would be activated at >700 nm and have better tumor specificities, which would reduce the generalized photosensitivities.[13] Newer photosensitizers in clinical trials include tin ethyl etiopurpurin (SnET2), monoL-aspartyl chlorin e6 (Npe6), benzoporphyrin derivative (BPD), and lutetium texaphyrin (Lu-Tex); these have absorption bands at 660, 664, 690 and 732 nm, respectively, and provoke only mild and transient skin photosensitivity. However, clinical studies using these photosensitizers are restricted to the treatment of cutaneous cancer,[14] early squamous cell carcinoma of the lung,[15] and recurrent prostate cancer;[16] therefore, full understanding of the relative merits of these sensitizers and approval for general clinical use are still pending. Figure 1 presents chemical structures for many of the photosensitizers discussed in this review.
2. Roles for Nanoparticle Delivery Systems in PDT The effectiveness of PDT is not only largely determined by the efficiency of singlet oxygen generation[17] upon light activation, but also the efficiency and selectivity to which therapeutic concentrations of the photosensitizer is delivered to the targeted tumor site with minimal uptake by non-targeted normal cells.[18] Incorporating PDT agents into nanoparticles can alter these properties to ultimately improve clinical outcome. Covalent conjugation or physical inclusion of photosensitizers to nanoparticle carrier systems can surround hydrophobic photosensitizers with a hydrophilic environment, improving their solubilities and preventing aggregation in blood. This process may also reduce the non-specific phototoxicity to the skin and eyes, enhance the antitumor efficacy,[19] and increase passive tumor accumulation via an enhanced permeability and retention (EPR) effect.[7] This will elevate levels of nanostructure-based photosensitizers either in proximity to the tumor tissue or inside the cells
S. H. Voon, Dr. L. V. Kiew Department of Pharmacology Faculty of Medicine University of Malaya 50603, Kuala Lumpur, Malaysia Dr. H. B. Lee, S. H. Lim, Dr. M. I. Noordin, Prof. L. Y. Chung Department of Pharmacy Faculty of Medicine University of Malaya 50603, Kuala Lumpur, Malaysia E-mail: [email protected]; [email protected] Dr. H. B. Lee, S. H. Lim Cancer Research Initiatives Foundation (CARIF) Subang Jaya Medical Center 47500, Subang Jaya, Selangor, Malaysia A. Kamkaew, Prof. K. Burgess Department of Chemistry Texas A&M University Box 30012, College Station, Texas 77842, USA DOI: 10.1002/smll.201401416
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In Vivo Studies of Photosensitizers for Cancer Therapy
after internalization. Majority of cellular internalization of nanoparticles occur through pinocytosis known as clathrinmediated endocytosis (CME).[20] The attachment of cancertargeting moieties such as folic acid,[21] F3 peptide[22] or RGD peptide[23] to the nano-drug carriers can further enhance the selectivity for tumors. Chemical modification of the structures of the nano-drug carriers may also confer alternate means for energy transfer to photosensitizers, which enables the use of deep tissue-penetrating light for the activation of the photosensitizers to treat deeply seated tumors.[24] The concept of subcellular targeted nanoparticles is being developed and only a limited number of strategies has been identified for endoplasmic reticulum, mitochondria and nucleus targeting. Further investigations are necessary to evaluate its impact and relevance for future clinical applications. The design of subcellular targeted nanoparticles with non-immunogenic stealth character, with the ability to overcome biological barriers and target site specificity, remains necessary.[20a] This review describes examples of how photosensitizers in nanostructures have advanced into the in vivo evaluation stage, focusing on the in vivo efficacies of the nanostructures as delivery agents and as energy transducers for photosensitizers in animal models (Table 1). We also summarize the in vivo characteristics of nanostructures in terms of biodegradability, clearance and side effect as well as the regulatory approval status for clinical use (Table 2). This article complements previous reviews on the design, synthesis, physicochemical properties, and in vitro therapeutic efficiencies of photosensitizers.[25]
Siew Hui Voon obtained her BSc in Biomedical Science from National University of Malaysia in 2009. She received a MyPhD scholarship from Ministry of Education Malaysia in 2012 to pursue her PhD under the guidance of Prof. Lip Yong Chung at the University of Malaya. Her current research of interest is in the field of nanomaterials for drug delivery in photodynamic cancer therapy.
Lik Voon Kiew obtained his doctorate in biopolymer technology and therapeutics from the University of Malaya, Malaysia. He is currently senior lecturer at the Pharmacology department, Faculty of Medicine of the University of Malaya, Malaysia. His current research interests include the development and in vitro/in vivo evaluation of targetable anticancer polymer therapeutics, renal targeting drug carriers, anticancer photodynamic therapeutics, and bioactive compounds for chronic disease treatment.
Lip Yong Chung received his doctorate in pharmacy from the University of Cardiff and joined Cardiff University as a research associate focusing on bioactive polymers and cell regeneration, and is currently at the Faculty of Medicine of the University of Malaya, Ma-
3. Liposomes
laysia as a Professor in Pharmaceutical Sciences. His current research interests include
Liposomes are spherical, closed membranes composed of concentric phospholipid bilayers with an aqueous inner compartment.[26] They can accommodate lipophilic photosensitizers by incorporating them into the lipid bilayer, which renders them hydrosoluble and prevents aggregation in an aqueous environment (Figure 2).[27] These structures naturally tend to accumulate in tumors, which enhances the ability of incorporated photosensitizers to target the tumor tissue.[28] Several studies have demonstrated a higher accumulation of photosensitizers and better PDT efficacy in tumor tissues for photosensitizer-containing liposomes compared to free photosensitizers.[29] Thus, the incorporation of Photofrin into liposomes resulted in 6 and 2.4-fold increases in in vivo tumoral accumulation for 9L gliosarcoma and U87 glioma nude mice models, respectively, compared to the free Photofrin control in a dextrose formulation. Tissue necrosis was significantly increased in both tumor types after PDT compared to the controls.[30] Enhanced tumor accumulation of hypocrellin A was also observed when a liposome-hypocrellin A nanoconstruct was tested in an S-180 sarcoma Kunming mouse model, with maximum accumulation in tumor at 12 h (0.48 µg/g) compared to the group treated with hypocrellin A-DMSO (0.14 µg/g).[31] This observation is consistent with the higher tumor regression at day 7 after PDT (relative regression percent of tumor, small 2014, DOI: 10.1002/smll.201401416
the discovery of anticancer and CNS active compounds, and development of nanodrug conjugates for therapy.
RRP = 87%) and with the PDT efficacy exhibited by the liposome-hypocrellin A-treated group relative to the hypocrellin A-DMSO treatment group (RRP = 14%). Metatetra(hydroxyphenyl) chlorin formulated in unilamellar dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol liposomal nanoconstructs has been applied to female Foxn1nu/nu mice implanted with EMT-6 mammary tumor cells. In the four month follow-up period, 79% of the primary tumors were cured and showed no sign of recurrence.[32] Visudyne, a commercially available liposomal formulation of benzoporphyrin derivative monoacid ring A (BPDMA) indicated for age-related macular degeneration[33] when applied to Meth-A sarcoma-bearing mice using antiangiogenic scheduling (short drug-light interval of 15 min) showed greater tumor suppression compared to conventional scheduling (longer drug-light interval of 3 h). In this, 40 and 60% of the tumor of Meth-A sarcoma-bearing mice were cured
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Figure 1. Some photosensitizers featured in this review.
at 0.25 and 0.5 mg/kg BPD-MA, respectively. These suggest Visudyne is effective in cancer PDT using antiangiogenic scheduling.[34] These liposomes of 70–100 nm led to an increase in passive accumulation in the tumors via the enhanced permeability and retention effect. However, the main drawback of conventional liposomes is that they are easily taken up by cells of the reticuloendothelial system after systemic administration. This results in their rapid removal from the blood into the liver and spleen, which in turn reduces the accumulation in the tumor tissue.[35] Some liposome modifications have been reported to counteract these delivery problems. For instance, polyethylene glycol has been added to liposomal surfaces to create more stable “sheathed” forms. Such PEGylation may protect liposomes from being recognized by opsonins and taken up by the reticuloendothelial system.[36] However, the increased stability provided by this type of
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modification of the liposomes may also decrease the liposome-cell interactions and reduce the transfer of the photosensitizer payload into tumor cells.[37]
4. Micelles Micelles are aggregates of surfactant molecules in liquid-colloid dispersions in which the hydrophilic head regions are in contact with surrounding solvent and the hydrophobic tails are in the center. The particle size range is normally within 5–100 nm. They are widely used to carry hydrophobic drugs, physically entrapped in or covalently bound to the hydrophobic core and delivered to tumors via passive or active targeting strategies.[38] Micelles can be classified according to the nature of the amphiphilic core: polymeric or lipid micelles. Polymeric micelles are formed from block copolymers,
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Table 1. In Vivo Studies Reported on Nanostructure-Based Photosensitizer Formulations. Nanostructure
Photosensitizer
Animal model
Photofrin (porfimer sodium); hyprocrellin A; metatetra(hydroxyphenyl) chlorin (mTHPC); Verteporfin
Athymic (nude) rats (strain Cr: NIH-rna) implanted with U87 glioma cells through craniectomy;[30a] male Kunming mice transplanted with S-180 sarcoma;[31] female Foxn1nu/nu mice injected subcutaneously with EMT6 cells;[32] male BALB/c mice injected subcutaneously with Meth-A sarcoma cells[34]
Polymer Micelles
Aluminum chloride phthalocyanine (AlClPc); protophorphyrin IX (PpIX)
Mice with squamous cell carcinoma (SCC7) tumor;[40] Balb/c mice implanted with EMT-6 tumors[41]
PEG-Lipid Micelles
Verteporfin; mesotetraphenylporphine (TPP)
Female C57BL/6 mice injected subcutaneously with LLC cells;[43a[ male DBA/2 mice implanted with rhabdomyosarcoma (M1) tumor cells[46]
Cremophor EL
Temocene; azabodipy
DBA/2 mice inoculated with the DBA/2 mastocytoma cell line P815;[53] Balb/c mice inoculated with the Balb/c colon adenocarcinoma cell line CT26.CL25 (ATCC, CRL-2639);[53] female Balb/c nu/nu mice injected with MDA-MB-231-GFP cells[51]
Poly(lactic-co-glycolic acid) (PLGA)
Zinc phthalocyanine; verteporfin
Female albino mice injected with Ehrlich ascites carcinoma cells;[56] male DBA/2 mice implanted with rhabdomyosarcoma (M1) tumor[18]
Dendrimers
Aminolevulinic acid (ALA); phthalocyanine
Male BALB/c mice injected with the LM3 cell line;[60] female nude mice (BALB/c nu/nu) subcutaneously transplanted with subcutaneous A549 tumor cells[61]
Chitosan
Protophorphyrin IX (PpIX); chlorin e6
Athymic nude mice inoculated with SCC7 cells;[65] athymic nude mice injected with HT-29 human colon adenocarcinoma cells[67,70[
LIPOPROTEIN NANOPARTICLES
Bacteriochlorin e6 bisoleate (Bchl-BOA)
Female athymic nude mice inoculated with HepG2 cells[77]
Silica
Protophorphyrin IX (PpIX); methylene blue; zinc phthalocyanine
Male athymic Nude-Foxn1nu mice subcutaneously implanted with glioblastoma multiforme;[82] female athymic Swiss nude mice and female athymic Naval Medical Research Institute nude mice injected subcutaneously with HCT 116 and A549 cells, respectively;[82] male athymic BALB/c (Balb/C-nu) mice inoculated subcutaneously with Hela cells;[80] female BALB/c nude mice implanted with H22 cells;[84] female Swiss nude mice xenografted with HCT-116 cells[85]
Gold
Silicon phthalocyanine 4; porphyrinbrucine conjugates; Chlorin e6
NuNu mice subcutaneously injected with basaloid squamous cell carcinoma PE/CA-PJ34 cells;[90a] mice with MDA-MB-435 tumor[92]
Calcium Phosphosilicate
Indocyanine green (ICG)
Female C3H/HeJ mice injected with 32D-p210-GFP cells[94]
Photofrin (porfimer sodium)
Male Fischer 344 rats implanted with rat 9L glioma cells[22]
N-(2-Hydroxypropyl)methacrylamide (HPMA)
meso-chlorin e6 monoethylene diamine (Mce6)
Female nu/nu mice with OVCAR-3 solid tumor[102b]
Liposome
Doxorubicin (Chemotherapeutic agent); Verteporfin
Female BALB/c mice inoculated subcutaneously with murine colon carcinoma Colo 26;[103] Male Copenhagen rats implanted subcutaneously and orthotopically with R3327-MatLyLu Dunning prostate tumor cells[104]
UPCONVERSION NANOPARTICLES (ENERGY TRANSDUCER)
Chlorin e6; merocyanine 540 (MC540); zinc phthalocyanine (ZnPc)
Female Balb/c mice injected subcutaneously with 4T1 cells;[24b] C57BL/6 female mice implanted subcutaneously with B16-F0 cells[21]
Titanium Dioxide
None
Nude mice injected subcutaneously with T-24 cells;[108] female BALB/c-nude mice inoculated with U87 cells[110]
Fullerenes (C60)
None
Female CDF1 mice inoculated with Meth A fibrosarcoma cells;[118a] female CDF1 mice inoculated with Meth AR1fibrosarcoma cells;[118b] male BALB/c mice inoculated with CT26-Luc cells;[122] mice xenografted with human-melanoma (COLO679)[123]
LIPOSOMES
MICELLES
POLYMER-BASED NANOPARTICLES
INORGANIC NANOPARTICLES
HYBRID NANOPARTICLES Polyacrylamide COMBINATION THERAPY (PDT & CHEMOTHERAPY)
NANOPARICLES WITH PHOTOSENSITIZING PROPERTIES
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Table 2. In Vivo Characteristics of Nanostructures and Their Regulatory Approval Status for Clinical Use. Nanostructure
Biodegradability
Metabolism & Clearance
Side Effects
FDA Approval Status for PDT use
Regulatory Approval Status for other anticancer drugs
LIPOSOMES
Yes
Vesicle opsonization by serum protein and subsequent uptake by the reticuloendothelial system; Complementmediated phagocytosis by Kupffer and endothelial cells of the liver as well as other phagocytic cells of the reticuloendothelial system[128]
NA
Yes
Clinically approved: Doxil (Doxorubicin) [US, EU][129]
Clinically approved: Visudyne (Verteporfin) to treat age-related macular degeneration
DaunoXome (Daunorubicin citrate) [US][129]
Phase I & II clinical trials: CGP 55847 (liposomal zinc(II)phthalocyanine) for cancer treatment[125]
Depocyt (Cytarabine) [US, EU][130] Mepact (Mifamurtide) [EU][131] Marqibo (Vincristine sulfate) [On market][132]
MICELLES Polymer Micelles
No
Degradation of polymer micelles, resulting in the formation of block copolymer unimers, which can be removed via renal excretion if the polymer chains are designed with a lower molecular weight than the critical value for renal filtration less than ∼20–40 kDa.[133]
Slow extravazation; Risk of chronic liver toxicity due to prolonged circulation and slower metabolism of drug which may exhibit toxic side effects[133]
No
Clinically approved: Taxotere (Docetaxel) [EU, US][129a] Phase II clinical trials: SP1049C (Pluronic block-copolymer doxorubicin)[129a,134]
PEG-Lipid Micelles
NA
NA
NA
No
NA
Cremophor EL
Slow
May be largely degraded in the blood compartment by serum carboxylesterase-induced degradation, causing a gradual release of the ricinoleic acid residues attached to the triglyceride structure; Hepatobiliary elimination; Less than 0.1% of administered dose via urinary excretion[52]
Associated with severe anaphylactoid hypersensitivity reactions, hyperlipidaemia, abnormal lipoprotein patterns, aggregation of erythrocytes and peripheral neuropathy[52]
No
Clinically approved: Taxol (Paclitaxel) [US][129a]
Cremophor EL
Slow
May be largely degraded in the blood compartment by serum carboxylesterase-induced degradation, causing a gradual release of the ricinoleic acid residues attached to the triglyceride structure; Hepatobiliary elimination; Less than 0.1% of administered dose via urinary excretion[52]
Associated with severe anaphylactoid hypersensitivity reactions, hyperlipidaemia, abnormal lipoprotein patterns, aggregation of erythrocytes and peripheral neuropathy[52]
No
Clinically approved: Taxol (Paclitaxel) [US][129a]
Undergo hydrolysis and biodegrades into lactic and glycolic acids. Lactic acid enter the tricarboxylic acid cycle and is metabolized and subsequently eliminated from the body as carbon dioxide and water. Glycolic acid is either excreted unchanged in the kidney or enters the tricarboxylic acid cycle and is eventually eliminated as carbon dioxide and water[55a]
NA
Approved by the US FDA for the use of drug delivery[135]
Phase II Clinical Trials: WST09 (TOOKAD) in Cremophor EL formulation[126a] Phase I Clinical Trias: Silicon Phthalocyanine Pc 4 in Cremophor EL formulation[126b]
POLYMER-BASED NANOPARTICLES Poly(lactic-coglycolic acid) (PLGA)
Yes
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NA
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Table 2. Continued Nanostructure
Biodegradability
Metabolism & Clearance
Side Effects
FDA Approval Status for PDT use
Regulatory Approval Status for other anticancer drugs
Dendrimers
NA
Renal clearance for dendrimers with diameter 3–10 nm[128]
NA
No
Phase III clinical trials: SH L 643A (Gadolinium) for diagnostic imaging[136]
Chitosan
Yes
Mainly degraded by lysozyme through the hydrolysis of the acetylated residues[137]
NA
No
NA
LIPOPROTEIN NANOPARTICLES
Yes
Catabolized by the endothelium-associated lipoprotein lipase, thereby generating free fatty acids, which are taken up by the liver, muscle, and adipose tissues[138]
NA
No
NA
Silica
Slow
Gradual biodegradation results in the formation of water-soluble salts of silicic acid, which are excreted by the kidney; Hepatobiliary excretion[139]
Caused granuloma formation in the organs of the reticulo-endothelial system, such as liver and spleen[139,140]
No
NA
Gold
No
Renal clearance for particles with diameter < 10 nm[128]
NA
No
Phase I clinical trials: AuroShell (gold nanoparticle) for laser therapy[141]
Calcium Phosphosilicate
No
Hepatobiliary clearance with minimal acute renal involvement[93a]
NA
No
NA
Yes
Phagocytosis by Kupffer cells followed by hepatobiliary and renal clearance[97]
NA
No
NA
N-(2-Hydroxypropyl) methacrylamide (HPMA)
No
Blood clearance by spleen macrophage and eliminated by glomerular filtration in the kidney[137]
NA
No
Phase II clinical trials: ProLindac (HPMA copolymer–DACH platinate)[142]
UPCONVERSION NANOPARTICLES (ENERGY TRANSDUCER)
No
Hepatobiliary clearance[24b]
NA
No
NA
Titanium Dioxide
No
94% and 2% of administered TiO2 was found in the liver and spleen and did not decrease up to 30 days after administration[143]
Can induce pathological lesions of the liver, spleen, kidneys, and brain[144]
No
NA
Fullerenes (C60)
No
Increased hepatobiliary accumulation and excrete via urinary tract[114,128]
NA
No
NA
INORGANIC NANOPARTICLES
HYBRID NANOPARTICLES Polyacrylamide
COMBINATION THERAPY (PDT & CHEMOTHERAPY)
NANOPARICLES WITH PHOTOSENSITIZING PROPERTIES
NA = not available
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Figure 2. Liposome. Size: 70–100 nm.
whereas lipid micelles are prepared from water-soluble polymers conjugated to lipids.
4.1. Polymer Micelles Polymer micelles are formed in an aqueous solution from amphiphilic block or graft copolymers consisting of hydrophilic and hydrophobic monomeric units, which form their corona and core, respectively (Figure 3a). These self-assembling structures have the ability to solubilize drugs with poor water solubility,[39] and uptake by the reticuloendothelial system is substantially reduced by the hydrophilic palisades of tethered polymer chains surrounding the drug-loaded core.[39d] The uptake of pH-responsive methoxy polyethylene glycol-poly(β-amino ester) block copolymer micelles (pHPMs) encapsulating protophorphyrin IX (PpIX) by the reticuloendothelial system has been shown to be reduced.[40] In SCC7 tumor-bearing mice, biodistribution evaluation measured by photon counts showed that the PpIX level in tumors for the PpIX-pH-PMs treated group were 10× higher than that of free PpIX treated group at 48 h post-injection. Ex vivo imaging of the excised organs (liver, lung, spleen, kidney, heart) and tumors showed the strongest fluorescence intensity in the tumors, while PpIX uptake in other normal organs was not significant except in liver and kidney, organs where PpIX is rapidly metabolized. In contrast, when free PpIX was delivered, strong fluorescent signals were observed mainly in the liver, whereas the tumor only presented a very weak fluorescent signal that could not be clearly distinguished from the body. In this study, complete ablation of tumors occurred in mice treated with PpIX-pH-PMs, whereas tumor growth continued in free PpIX-treated group. Histological examinations revealed that most of the tumor cells in the mice treated with PpIX-pH-PMs were severely damaged or destroyed at day 10 after treatment, while incomplete tumor cell death was observed in the mice treated with free PpIX. N-iso-Propylacrylamide (NIPAM) copolymer micelles have been co-polymerized with a pH-sensitive moiety, methacrylic acid and N-vinyl-2-pyrrolidone units to impart
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Figure 3. (a) Polymer micelle. Size: 30–122 nm; (b) PEG-lipid micelles. Size 13–30 nm; (c) Cremophor EL. Size: 30 nm.
tumor-targeting properties and stealth properties, respectively.[41] The methacrylic acid is believed to increase tumoral localization, while the N-vinyl-2-pyrrolidone provides a nonionic hydrophilic shield to reduce uptake by the mononuclear phagocytes, thus increasing the time the micelles remain circulating in the blood. When loaded with aluminum chloride phthalocyanine (AlClPc), these AlClPc-polymer micelle formulations did not show increased tumoral uptake of AlClPc over the Cremophor® EL control formulation (1.8–2% vs. 2.4% AlClPc of injected dose/g, respectively). Both formulations showed similar PDT efficacy with no tumor recurrence in 80% of mice bearing intradermal EMT-6 tumors. However, the low toxicity of these modified NIPAM polymeric micelles and their strong affinity for AlClPc makes them a good alternative to Cremophor EL for the administration of poorly water-soluble phthalocyanines for PDT.
4.2. Polyethylene Glycol–Lipid Micelles Polyethylene glycol-lipid micelles consist of polyethylene glycol conjugated to a relatively short but hydrophobic diacyl phospholipid moiety such as phosphatidyl ethanolamine (Figure 3b).[42] In this construct, the phosphatidyl ethanolamine binds to the hydrophobic photosensitizers[43] and the
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polyethylene glycol prevents the rapid uptake of the micelle nanoparticles by the reticuloendothelial system.[44] Roby et al.[43a] modified the polyethylene glycol-lipid micelles by attaching antibodies to the micellar surface as tumor-targeting ligands for meso-tetraphenylporphine (TPP) delivery. Specifically, they used the monoclonal antibody 2C5 (MAb 2C5) to target surface-bound nucleosomes, which are released from apoptotically dying cancer cells. Two-tothree-fold improved accumulation of TPP in tumors was achieved compared to the non-targeted polyethylene glycolphosphatidyl ethanolamine micelles in female C57BL/6 mice injected subcutaneously with Lewis lung carcinoma cells. The antibody-targeted micelles resulted in complete inhibition of tumor growth until day 35 after PDT treatment, while those lacking the antibody only reduced tumor growth by 50%. Treatment with free TPP only caused slight tumor growth suppression compared to the untreated control. Histological examination of the tumors showed significantly higher tumor cell death in the mice treated with TPP-loaded MAb 2C5-polyethylene glycol-phosphatidyl ethanolamine-immunomicelles. Formulation of benzoporphyrin photosensitizers in methoxypoly(ethylene glycol)-distearoylphosphatidylethanolamine (mPEG-DSPE) micelles also increased their solubility.[45] An intravenous dose of 1.4 µmol/kg of an mPEG2000-DSPE micellar formulation of an A-ring benzoporphyrin derivative at a 6:1 lipid/drug ratio caused tumor regression in male DBA/2 mice implanted with rhadbomyosarcoma (M1) tumor cells. The mice remained free of tumors up to 20 days after PDT, while there was no observable tumor reduction in the control mice.[46]
4.3. Cremophor EL Cremophor EL is a self-assembling micelle containing glycerol polyethylene glycol ricinoleate used to solubilize hydrophobic photosensitizing agents and promote their distribution into plasma lipoproteins (Figure 3c).[47] It has been used in combination with ethanol to solubilize lipophilic photosensitizers, such as TOOKAD,[48] phthalocyanine,[49] purpurin[50] and azabodipy-based photosensitizers.[51] The in vivo use of Cremophor EL has been associated with anaphylactic hypersensitivity reaction and neurotoxicity,[52] but it is widely used in early preclinical studies because it conveniently and efficiently emulsifies lipophilic entities. Temocene incorporated in Cremophor EL micelles showed a better in vivo response than free temocene. Total regression of the principal tumors and remission of tumors in Balb/c mice subcutaneously inoculated with CT26.CL25 tumor cells and DBA/2 mice inoculated with mastocytoma cells P815 for 40 and 60 days, respectively, were observed.[53] The best PDT response was obtained if the photoactivation was carried out after a short drug-light interval of 15 min after injection of the temocene-loaded micelles. Tumor regression was due to tumor-associated vascular damage leading to tumor hypoxia. The kinetics of the drug accumulation suggests that the small Cremophor EL micellar size (30 nm) confers fast tumoral accumulation, and the micelles are small 2014, DOI: 10.1002/smll.201401416
then rapidly cleared by the reticuloendothelial system. This temocene-loaded Cremophor EL micellar formulation significantly improves the PDT effect of temocene and is particularly suited for vascular-targeted treatments. Cremophor EL micelles were also used for the delivery of a lead BF2-azadipyrromethene molecule, ADMP06, targeting the mammary tumor vasculature[51] and were evaluated in nude mice inoculated subcutaneously with MDA-MB-231-GFP cells. After PDT treatment, tumor ablation was demonstrated for 71% of the treated nude mice with no recurrence for 6 months.
5. Polymer-based Nanoparticles Some polymer-based nanoparticles can have good aqueous dispersions, high drug-loading capacities, and extended release properties. Modifications are possible to alter their particle size, surface characteristics, and ability to achieve active or passive targeting.[54] Several categories of polymeric nanoparticles have been studied as carriers for PDT agents, including synthetic polymers e.g., poly(lactide-co-glycolide) copolymers (PLGA) and dendrimers, and natural polymers such as chitosan.
5.1. Poly(lactic-co-Glycolic Acid) (PLGA) PLGAs are copolymers of lactic and glycolic acids. These structures can have excellent biocompatibility, biodegradability and mechanical strength. They have been formulated into various delivery systems for carrying a variety of active agents such as vaccines, peptides, proteins, and macromolecules (Figure 4a). Some of these agents are now approved by the US Food and Drug Administration for use in drug delivery.[55] Fadel et al.[56] formulated PLGA nanoparticles to enhance tissue uptake, permeation, and targeting of zinc (II) phthalocyanine for photodynamic therapy. Female albino mice implanted with Ehrlich ascites carcinoma cells and treated with nanoparticle formulated-zinc (II) phthalocyanine survived significantly longer than those treated with free drug (mean = 60 and 25 days, respectively), while the mean survival time of an untreated control group was 15 days. Two weeks after the treatment, the mean tumor volumes for the nanoparticle formulated-zinc (II) phthalocyanine group were found to be 1.5 times smaller than those in animals treated with the free drug, and the animals in the former group exhibited a longer tumor growth delay of 39 days. PLGA nanoparticles have also been used to carry Verteporfin. Free Verteporfin causes extensive skin photosensitivity,[57] but male DBA/2 mice treated with Verteporfin loaded in PLGA nanoparticles showed only a very mild and short period of skin photosensitivity as measured by erythema/eschar formation and edema observations 24 h after PDT in a rhabdomyosarcoma (M1) tumor model.[18] Total tumor eradication with no tumor regrowth was observed up to 14 days after PDT and on day 20, only 34% of the mice showed tumor regrowth.
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of the dendrimer-conjugate for optimum cellular uptake. Many dendrimers also have good tissue biodistribution properties.[59] Casas et al.[60] used ester coupling reactions to synthesize a dendrimer bearing eighteen aminolevulinic acid residues (18m-ALA). This agent induced sustained porphyrin production that peaked at 24 h in male Balb/c mice in contrast to the response to free aminolevulinic acid, which peaked sharply at 3 to 4 h. The tumoral accumulation of 18m-ALA was 7.5 times higher for the 18m-ALA dendrimer than for free aminolevulinic acid at the same concentration. However, the in vivo efficacy was not determined. Use of polyionic micelles has been studied to explore the hypothesis that they could reduce π–π interactions of photosensitizers that tend to lead to aggregation.[61] Thus dendrimeric phthalocyanine complexed with poly-L-lysinepolyethylene glycol polymeric micelles (DPc/m) were evaluated in female Balb/cnu/nu mice subcutaneously implanted in the back with human lung adenocarcinoma A549 cells.[61] A significantly reduced tumor growth rate was observed for the mice treated with DPc/m compared to the dendrimeric phthalocyanine-treated and control (without drug treatment) groups. Moreover, the PDT effect of DPc/m was significantly greater than Photofrin alone, even though the injected dose of DPc/m, based on the photosensitizing units, was 7.3-fold lower than Photofrin. DPc/m did not induce phototoxicity after PDT treatment, whereas severe skin and liver damage was observed in Photofrin-treated mice.
5.3. Chitosan Nanoparticles
Figure 4. (a) Poly(lactic-co-glycolic acid) (PLGA) nanoparticle. Size: 117–450 nm; (b) Dendrimer. Size: 50 nm; (c) Chitosan nanoparticle. Size: 260–350 nm.
5.2. Dendrimers Dendrimers are regularly branched, three-dimensional structures composed of a central core molecule and branching units that can be conjugated to drug molecules (Figure 4b).[58] Dendritic branching terminals may be functionalized with solubilizing groups, targeting groups or other moieties. Dendrimers have the advantages of high drug payload and the ability to control and modify the size and lipophilicity
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Synthetic polymers are easily modified to achieve desirable properties, but natural polymers are of interest because they are biodegradable and readily available. Chitosan, a product of partial deacetylation of the natural polysaccharide chitin, is the second most abundant biopolymer after cellulose. This natural polysaccharide has been widely used in various biomedical and pharmaceutical applications,[62] due to its wide availability, bioavailability, low immunogenicity and suitability for chemical modifications (Figure 4c).[63] Glycol chitosan, a chitosan derivative with ethylene glycol groups added to its backbone to enhance its water solubility,[64] was used by Lee et al.[65] to physically encapsulate protophorphyrin IX in nanoparticles. In vivo tumor uptake of the protophorphyrin IX-loaded nanoparticles was 2.3× higher than free protophorphyrin IX in squamous cell carcinoma (SCC7)bearing athymic nude mice. Moreover, the total fluorescent photon counts per organ for the tumor tissues were 1.49 to 3.8fold higher than in other organs, implying favorable biodistribution properties. On day 14 after treatment, measurements of the tumor volumes showed a 44% reduction in the protophorphyrin IX-loaded nanoparticle-treated group, where the same regimen of free protophorphyrin IX did not produce substantial tumor volume reduction. Furthermore, histological examination showed that most of the tumor cells were severely damaged or destroyed in the mice treated with the protophorphyrin IX-loaded nanoparticles, whereas incomplete tumor death was observed in the mice treated with free protophorphyrin IX.
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Nanoparticles in which the cargo is non-covalently incorporated can exhibit burst release profiles; such sudden liberation of cargo can reduce tumor targeting and increase damage to normal tissues.[66] To circumvent this, Lee et al.[67] used chemical conjugation rather than physical loading to prepare protophorphyrin IX-conjugated glycol chitosan nanoparticles. These particles exhibited desirable “safety catch” photodynamic properties because the highly dense protophorphyrin IX cores remain in a quenched ‘off’ state until they are taken up into cells; in their off-state they may not generate sufficient singlet oxygen to produce phototoxicity.[68] After cellular uptake, the dense protophorphyrin IX cores disintegrate in the intracellular environment,[69] permitting the protophorphyrin IX to revert to a photocytotoxic ‘on’ state. Thus, in mice bearing the HT-29 human colon adenocarcinoma, the photon counts in the tumor tissues of mice treated with the protophorphyrin IX-conjugated glycol chitosan nanoparticles were 1.4 or 2.1-fold higher than in the tumors of mice treated with protophorphyrin IX physically loaded into glycol chitosan nanoparticles and free protophorphyrin IX, respectively. The tumor sizes were at least 5.2-fold smaller in the mice treated with the protophorphyrin IX-conjugated glycol chitosan nanoparticles than in those treated with free protophorphyrin IX. Furthermore, histological examination revealed that little apoptosis and necrosis was observed in the tumors of mice treated with free protophorphyrin IX, in contrast to the large amount of cell death in the tumor tissues of those treated with the protophorphyrin IX-conjugated glycol chitosan nanoparticles. Lee et al.[70] supported his findings above by comparing the antitumor efficacy of chlorin e6 of chemically conjugated and physically loaded glycol-chitosan nanoparticles in HT-29 human colon adenocarcinoma tumor-bearing mice. Severe photo-induced tumor necrosis was induced in the mice treated with the conjugated chlorin e6-glycol-chitosan nanoparticles whereas the group treated with the physically loaded chlorin e6-glycol-chitosan nanoparticles failed to show noticeable phototoxicity in the tumor tissues. Final tumor volumes in the mice treated with the conjugated chlorin e6-glycol-chitosan-nanoparticles were approximately 160 mm3 at 20 days post-injection, which was significantly smaller than the size of the tumors (approximately 560 mm3) in the mice treated with the physically loaded chlorin e6-glycol-chitosan-nanoparticles. The improved antitumor efficacy of conjugated chlorin e6-glycol-chitosan nanoparticles in these experiments was attributed to more efficient accumulation of these particles in the tumors.[70] The fluorescence of the conjugated chlorin e6-glycolchitosan-nanoparticles in the tumor tissues persisted for two days after administration. In contrast, the physically loaded chlorin e6-glycol-chitosan nanoparticles showed no specific tumor accumulation: instead, an intense whole-body fluorescence at 3 h post-injection was followed by a rapid decrease in fluorescence. Furthermore, the total photon counts of the chemically conjugated chlorin e6-glycol-chitosan nanoparticles in tumor tissue were approximately 6 to 7-fold higher than that of the physically loaded chlorin e6-glycolchitosan nanoparticles. small 2014, DOI: 10.1002/smll.201401416
6. Lipoprotein Nanoparticles Lipoprotein nanoparticles are composed of naturally occurring apoproteins, phospholipids and cholesterol on surfaces that encapsulate cholesterol esters and triglycerides in the hydrophobic core.[71] They are potentially excellent cancer drug delivery systems because they can remain in circulation for an extended period while evading the reticuloendothelial system.[72] The hydrophobic core facilitates incorporation of poorly soluble photosensitizers, and these materials are amenable to various drug-linking strategies (Figure 5).[71] Low-density-lipoprotein (LDL) (60 days, and no tumors were present in two of the three rats after 6 months.
adriamycin to nude mice with human epithelial ovarian carcinoma. The HPMA copolymer-adriamycin conjugate with 2.2 mg/kg adriamycin equivalent plus HPMA copolymermeso-chlorin e6 at 1.5 and 8.7 mg/kg meso-chlorin e6 equivalent with light resulted in significant tumor regression relative to receiving either agent and/or with light alone. No shock syndrome was observed in the mice; this can result from massive agent-stimulated prostaglandin release, which causes platelet aggregation and damage and leads to obstruction of blood flow. This study suggests that an HPMA copolymer carrier can extend the safety margin of meso-chlorin e6 in vivo in PDT because free meso-chlorin e6 at between 2.5 and 10 mg/kg not only caused tumor regression but also caused shock syndrome. Synder et al. developed a combination therapy using PDT to enhance the delivery and efficacy of macromolecule-based cancer drug such as Doxil a pegylated liposome encapsulated doxorubicin with an average diameter of 100 nm.[103] Prior to the administration of Doxil, murine Colo 26 tumor-bearing mice was treated with the photosensitizer 2-[1-hexyloxyethyl]2-devinyl pyropheophorbide-a (HPPH) at 0.4 mol/kg and subjected to PDT at 48 J/cm2 and 14 mW/cm2. This increased the accumulation of doxorubicin in transplanted Colo 26 tumors significantly without concomitant enhancement of systemic or local toxicity. Eighty percent of tumor-bearing mice treated with the combination of PDT and Doxil at 5 mg/kg were cured compared to the untreated tumor-bearing control mice. On the other hand, a dose of just 5 mg/kg Doxil without PDT only showed modest tumor growth delay without complete remission. Furthermore, the combination of PDT and Doxil treatment also reduced skin toxicity in terms of erythema, broken vessels and light eschar formation. This phenomenon is supported by Bin Chen et al.’s findings in relation to subcutaneous and orthotopic MatLyLu rat prostate models, where tumor uptake of macromolecules such as Evans blue-albumin and high molecular weight FITC-labeled dextran was significantly increased following PDT treatment. This suggests that PDT increased vascular barrier dysfunction in the MatLyLu tumors. The observation on the adherence of blood cells to vessel wall shortly after PDT further supports the loss of endothelial integrity in the blood vessels.[104]
9. Combination Therapy (PDT and Chemotherapy) N-(2-Hydropropyl)methacrylamide (HPMA) is a water-soluble, biocompatible polymeric drug carrier that can be easily formulated to include targeting ligands[100] and has been shown to preferentially accumulate in tumors via the enhanced permeability and retention effect.[101] HPMA copolymer-bound drugs have been developed for the combination of PDT and chemotherapy (Figure 8).[102] Peterson et al.[102a] designed a combination of two HPMA nanoparticle systems to co-deliver meso-chlorin e6 and
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Figure 8. Combination therapy. Size: 25 nm. © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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10. Upconverting Nanoparticles (Energy Transducers) One way to overcome the limitations imposed by poor tissue penetration of shorter wavelength (
