Photosensitizer Protoporphyrin IX Research Articles

Mesoporous silica coated spicules for photodynamic therapy of metastatic melanoma

We report on the fabrication of mesoporous silicon dioxide coated Haliclona sp. spicules (mSHS) to enhance the delivery of the insoluble photosensitizer protoporphyrin IX (PpIX) into deep skin layers and mediate photodynamic therapy for metastatic melanoma in mice. The mSHS are dispersed sharp edged and rod-like micro-particles with a length of approximate 143.6 ± 6.4 μm and a specific surface area of 14.9 ± 3.4 m2/g. The mSHS can be topically applied to the skin, adapting to any desired skin area and lesion site. The insoluble PpIX were incorporated into the mesoporous silica coating layers of mSHS (mSHS@PpIX) with the maximum PpIX loading capacity of 120.3 ± 3.8 μg/mg. The mSHS@PpIX significantly enhanced the deposition of PpIX in the viable epidermis (5.1 ± 0.4 μg/cm2) and in the dermis (0.5 ± 0.2 μg/cm2), which was 154 ± 11-fold and 22 ± tenfold higher than those achieved by SHS, respectively. Topical delivery of PpIX using mSHS (mSHS@PpIX) completely eradicated the primary melanoma in mice in 10 days without recurrence or metastasis over 60 days. These results demonstrate that mSHS can be a promising topical drug delivery platform for the treatment of diverse cutaneous diseases, such as metastatic melanoma.Graphic

Mitochondrial targeting improves the selectivity of singlet-oxygen cleavable prodrugs in NMIBC treatment.

Mitochondria play an essential role in cancer treatment by providing apoptotic signals. Hexyl aminolevulinate, an FDA-approved diagnosis for non-muscle invasive bladder cancer, induces the production of protoporphyrin IX (PpIX) preferentially by mitochondria in cancer cells. Photosensitizer PpIX upon illumination can release active chemotherapy drugs from singlet oxygen-activatable prodrugs. Prodrugs placed close enough to PpIX formed in mitochondria can improve the antitumor efficiency of PpIX-PDT. The preferred uptake of prodrugs by cancer cells and tumors can further enhance the selective damage of cancer cells over non-cancer cells and surrounding normal tissues. Mitochondriotropic prodrugs of anticancer drugs, such as paclitaxel and SN-38, were synthesized using rhodamine, a mitochondrial-targeting moiety. Invitro, the mitochondrial targeting helped achieve preferential cellular uptake in cancer cells. In RT112 cells (human bladder cancer cells), intracellular prodrug concentrations were 2-3 times higher than the intracellular prodrug concentrations in BdEC cells (human bladder epithelial cells), after 2 h incubation. In an orthotopic rat bladder tumor model, mitochondria-targeted prodrugs achieved as much as 34 times higher prodrug diffusion in the tumor area compared to the nontumor bladder area. Overall, mitochondria targeting made prodrugs more effective in targeting cancer cells and tumors over non-tumor areas, thereby reducing nonspecific toxicity.

Theranostic Uses of the Heme Pathway in Neuro-Oncology: Protoporphyrin IX (PpIX) and Its Journey from Photodynamic Therapy (PDT) through Photodynamic Diagnosis (PDD) to Sonodynamic Therapy (SDT).

ALA PDT, first approved as a topical therapy to treat precancerous skin lesions in 1999, targets the heme pathway selectively in cancers. When provided with excess ALA, the fluorescent photosensitizer PpIX accumulates primarily in cancer tissue, and ALA PDD is used to identify bladder and brain cancers as a visual aid for surgical resection. ALA PDT has shown promising anecdotal clinical results in recurrent glioblastoma multiforme. ALA SDT represents a noninvasive way to activate ALA PDT and has the potential to achieve clinical success in the treatment of both intracranial and extracranial cancers. This review describes the creation and evolution of ALA PDT, from the treatment of skin cancers to PDD and PDT of malignant brain tumors and, most recently, into a noninvasive form of PDT, ALA SDT. Current clinical trials of ALA SDT for recurrent glioblastoma and high-grade gliomas in adults, and the first pediatric ALA SDT clinical trial for a lethal brainstem cancer, diffuse intrinsic pontine glioma (DIPG), are also described.

Regulating Photosensitizer Metabolism with DNAzyme-Loaded Nanoparticles for Amplified Mitochondria-Targeting Photodynamic Immunotherapy.

Mitochondria-specific photosensitizer accumulation is highly recommended for photodynamic therapy and mitochondrial DNA (mtDNA) oxidative damage-based innate immunotherapy but remains challenging. 5-Aminolevulinic acid (ALA), precursor of photosensitizer protoporphyrin IX (PpIX), can induce the exclusive biosynthesis of PpIX in mitochondria. Nevertheless, its photodynamic effect is limited by the intracellular biotransformation of ALA in tumors. Here, we report a photosensitizer metabolism-regulating strategy using ALA/DNAzyme-co-loaded nanoparticles (ALA&Dz@ZIF-PEG) for mitochondria-targeting photodynamic immunotherapy. The zeolitic imidazolate framework (ZIF-8) nanoparticles can be disassembled and release large amounts of zinc ions (Zn2+) within tumor cells. Notably, Zn2+ can relieve tumor hypoxia for promoting the conversion of ALA to PpIX. Moreover, Zn2+ acts as a cofactor of rationally designed DNAzyme for silencing excessive ferrochelatase (FECH; which catalyzes PpIX into photoinactive Heme), cooperatively promoting the exclusive accumulation of PpIX in mitochondria via the "open source and reduced expenditure" manner. Subsequently, the photodynamic effects derived from PpIX lead to the damage and release of mtDNA and activate the innate immune response. In addition, the released Zn2+ further enhances the mtDNA/cGAS-STING pathway mediated innate immunity. The ALA&Dz@ZIF-PEG system induced 3 times more PpIX accumulation than ALA-loaded liposome, significantly enhancing tumor regression in xenograft tumor models.

Continuous Spatiotemporal Therapy of A Full-API Nanodrug via Multi-Step Tandem Endogenous Biosynthesis

Nanomedicine holds great promise to enhance cancer therapy. However, low active pharmaceutical ingredient (API) loading content, unpredictable drug release, and potential toxicity from excipients limit their translational capability. We herein report a full-API nanodrug composed of FDA-approved 5-aminolevulinic acid (ALA), human essential element Fe3+, and natural bioactive compound curcumin with an ideal API content and pH-responsive release profile for continuous spatiotemporal cancer therapy achieved by multi-step tandem endogenous biosynthesis. First, ALA enzymatically converts into photosensitizer protoporphyrin IX (PpIX). Afterward, multiple downstream products including carbon monoxide (CO), Fe2+, biliverdin (BV), and bilirubin (BR) are individually biosynthesized through the PpIX-heme-CO/Fe2+/BV-BR metabolic pathway, further cooperating with released Fe3+ and curcumin, ultimately eliciting mitochondria damage, membrane disruption, and intracytoplasmic injury. This work not only provides a paradigm for exploiting diversified metabolites for tumor suppression, but also presents a safe and efficient full-API nanodrug, facilitating the practical translation of nanodrugs.

Photodynamic Effects with 5-Aminolevulinic Acid on Cytokines and Exosomes in Human Peripheral Blood Mononuclear Cells from Patients with Crohn's Disease.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) which is the precursor of the photosensitizer protoporphyrin IX (PpIX) is an available treatment for several diseases. ALA-PDT induces the apoptosis and necrosis of target lesions. We have recently reported the effects of ALA-PDT on cytokines and exosomes of human healthy peripheral blood mononuclear cells (PBMCs). This study has investigated the ALA-PDT-mediated effects on PBMC subsets from patients with active Crohn's disease (CD). No effects on lymphocyte survival after ALA-PDT were observed, although the survival of CD3-/CD19+ B-cells seemed slightly reduced in some samples. Interestingly, ALA-PDT clearly killed monocytes. The subcellular levels of cytokines and exosomes associated with inflammation were widely downregulated, which is consistent with our previous findings in PBMCs from healthy human subjects. These results suggest that ALA-PDT may be a potential treatment candidate for CD and other immune-mediated diseases.

Radiation-induced photodynamic therapy using calcium tungstate nanoparticles and 5-aminolevulinic acid prodrug.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) prodrug is a clinically tried and proven treatment modality for surface-level lesions. However, its use for deep-seated tumors has been limited due to the poor penetration depth of visible light needed to activate the photosensitizer protoporphyrin IX (PPIX), which is produced from ALA metabolism. Herein, we report the usage of poly(ethylene glycol-b-lactic acid) (PEG-PLA)-encapsulated calcium tungstate (CaWO4, CWO for short) nanoparticles (PEG-PLA/CWO NPs) as energy transducers for X-ray-activated PDT using ALA. Owing to the spectral overlap between radioluminescence afforded by the CWO core and the absorbance of PPIX, these NPs can serve as an in situ visible light activation source during radiotherapy (RT), thereby mitigating the limitation of penetration depth. We demonstrate that this effect is observed across different cell lines with varying radio-sensitivity. Importantly, both PPIX and PEG-PLA/CWO NPs exhibit no significant toxicities at therapeutic doses in the absence of radiation. To assess the efficacy of this approach, we conducted a study using a syngeneic mouse model subcutaneously implanted with inherently radio-resistant 4T1 tumors. The results show a significantly improved prognosis compared to conventional RT, even with as few as 2 fractions of 4 Gy X-rays. Taken together, these results suggest that PEG-PLA/CWO NPs are promising agents for application of ALA-PDT in deep-seated tumors, thereby significantly expanding the utility of the already established treatment strategy.

Multifunctional Protein Hybrid Nanoplatform for Synergetic Photodynamic-Chemotherapy of Malignant Carcinoma by Homologous Targeting Combined with Oxygen Transport.

Photodynamic therapy (PDT) under hypoxic conditions and drug resistance in chemotherapy are perplexing problems in anti-tumor treatment. In addition, central nervous system neoplasm-targeted nanoplatforms are urgently required. To address these issues, a new multi-functional protein hybrid nanoplatform is designed, consisting of transferrin (TFR) as the multicategory solid tumor recognizer and hemoglobin for oxygen supply (ODP-TH). This protein hybrid framework encapsulates the photosensitizer protoporphyrin IX (PpIX) and chemotherapeutic agent doxorubicin (Dox), which are attached by a glutathione-responsive disulfide bond. Mechanistically, ODP-TH crosses the blood-brain barrier (BBB) and specifically aggregated in hypoxic tumors via protein homology recognition. Oxygen and encapsulated drugs ultimately promote a therapeutic effect by down-regulating the abundance of multidrug resistance gene 1 (MDR1) and hypoxia-inducible factor-1-α (HIF-1α). The results reveal that ODP-TH achieves oxygen transport and protein homology recognition in the hypoxic tumor occupation. Indeed, compared with traditional photodynamic chemotherapy, ODP-TH achieves a more efficient tumor-inhibiting effect. This study not only overcomes the hypoxia-related inhibition in combination therapy by targeted oxygen transport but also achieves an effective treatment of multiple tumors, such as breast cancer and glioma, providing a new concept for the construction of a promising multi-functional targeted and intensive anti-tumor nanoplatform.

Effect of a 5-aminolevulinic acid gel and 660 nm red LED light on human oral osteoblasts: a preliminary in vitro study

This study aimed to evaluate the effects of a new photodynamic protocol (ALAD-PDT) on primary human osteoblasts (hOBs). The ALAD-PDT protocol consists of a heat-sensitive gel with 5% 5-delta aminolevulinic acid commercialized as Aladent (ALAD), combined with 630 nm LED. For this purpose, the hOBs, explanted from human mandible bone fragments, were used and treated with different ALAD concentrations (10%, 50%, 100% v/v) incubated for 45 min and immediately afterwards irradiated with a 630 nm LED device for 7 min. The untreated and unirradiated cells were considered control (CTRL). The cellular accumulation of the photosensitizer protoporphyrin IX (PpIX), the proliferation, the alkaline phosphatase (ALP) activity, and the calcium deposition were assessed. All concentrations (10, 50, 100%) determined a significant increment of PpIX immediately after 45 min of incubation (0 h) with the highest peak by ALAD (100%). The consequent 7 min of light irradiation caused a slight decrease in PpIX. At 48 h and 72 h, any increment of PpIX was observed. The concentration 100% associated with LED significantly increased hOB proliferation at 48 h (+ 46.83%) and 72 h (+ 127.75%). The 50% and 100% concentrations in combination to the red light also stimulated the ALP activity, + 12.910% and + 14.014% respectively. The concentration 100% with and without LED was selected for the assessment of calcium deposition. After LED irradiation, a significant increase in calcium deposition was observed and quantified (+ 72.33%). In conclusion, the ALAD-PDT enhanced proliferation, the ALP activity, and mineralized deposition of human oral osteoblasts, highlighting a promising potential for bone tissue regeneration.

5-Aminolevulinic Acid-Hyaluronic Acid Complexes Enhance Skin Retention of 5-Aminolevulinic Acid and Therapeutic Efficacy in the Treatment of Hypertrophic Scar.

Hypertrophic scar is a serious skin disorder, which reduces the patient's quality of life. 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy has been used to treat patients with hypertrophic scar. However, the poor skin retention of 5-ALA limited the therapeutic effect. In this study, we constructed the 5-ALA-hyaluronic acid (HA) complex to potentially prolong the skin retention of 5-ALA for improving the therapeutic efficacy. HA is a polysaccharide with viscoelasticity and the carboxyl groups could conjugate with amino groups of 5-ALA via electrostatic interaction. The protoporphyrin IX (PpIX) assay revealed that 5-ALA-HA complexes markedly enhanced the skin retention, resulting in increased generation and accumulation of endogenous photosensitizer PpIX. Furthermore, 5-ALA-HA complexes allowed PpIX to be maintained at a high level for 12h, much longer than the 3h of 5-ALA alone. And then, the accumulative PpIX induced by 5-ALA-HA in human hypertrophic scar fibroblasts (HSF) was triggered by laser irradiation to produce sufficient reactive oxygen species, leading to efficient necrosis and apoptosis of HSF. In vivo therapeutic efficacy study indicated that 5-ALA-HA effectivelyreducedthe appearance and scar thickness, and the scar elevation index with 5-ALA-HA treatment was significantly lower than other groups, suggesting that the 5-ALA-HA-treated scar became flattened and was closely matched to the unwounded tissues. Moreover, 5-ALA-HA treatment markedly downregulated the gene expression levels of α-SMA and TGF-β1, demonstrating attenuated the scar formation and growth. Therefore, the 5-ALA-HA complex enhancing skin retention and PpIX accumulation at the lesion site provide a promising therapeutic strategy for hypertrophic scar.

Identification and pharmacological modification of resistance mechanisms to protoporphyrin-mediated photodynamic therapy in human cutaneous squamous cell carcinoma cell lines

BackgroundPhotodynamic therapy (PDT) is clinically approved to treat neoplastic skin diseases such as precursors of cutaneous squamous cell carcinoma (cSCC). In PDT, 5-aminolevulinic acid (5-ALA) drives the selective formation of the endogenous photosensitizer protoporphyrin IX (PpIX). Although 5-ALA PDT is clinically highly effective, resistance might occur due to decreased accumulation of PpIX in certain tumors. Such resistance may be caused by any fundamental step of PpIX accumulation: 5-ALA uptake, PpIX synthesis and PpIX efflux. MethodsWe investigated PpIX accumulation and photodynamically induced cell death in PDT refractory SCC-13, PDT susceptible A431, and normal human epidermal keratinocytes (NHEK). Expression of genes associated with cellular PpIX kinetics was investigated on mRNA and protein level. PpIX accumulation and cell death upon illumination were pharmacologically manipulated using drugs targeting 5-ALA uptake, PpIX synthesis or efflux. ResultsThe experiments indicate that taurine transporter (SLC6A6) is the major pathway for 5-ALA uptake in cSCC cells, while being less important in NHEK. Downregulation of PpIX synthesis enzymes in SCC-13 was counteracted by methotrexate (MTX) treatment, which restored PpIX formation and cell death. PpIX efflux inhibitors targeting ABC transporters led to significantly increased PpIX accumulation in SCC-13, thereby fully overcoming resistance. ConclusionsThe results indicate a conserved threshold for PpIX accumulation with respect to PDT-resistance. Cells showed increased viability after PDT at PpIX concentrations below 1.5 nM. Selective uptake of 5-ALA via taurine transporter SLC6A6 in cutaneous tumor cells is novel but unrelated to resistance. MTX can partially abrogate resistance by PpIX synthesis enzyme induction, while efflux mechanisms via ABC transporters seem the main driving force and promising drug targets.

An Intracellular Self-Assembly-Driven Uninterrupted ROS Generator Augments 5-Aminolevulinic-Acid-Based Tumor Therapy.

Free radical therapy based on 5-aminolevulinic acid (ALA, a precursor of the photosensitizer protoporphyrin IX (PpIX)) has been approved by the US Food and Drug Administration for clinical tumor treatment. However, PpIX can be quickly converted into photoinactive heme, leading to unexpectedly paused production of free radicals and severely hindering its therapeutic benefits. Here, inspired by the natural biotransformation of ALA (ALA-PpIX-heme), an uninterrupted reactive oxygen species generator (URG) that converts useless heme to peroxidase mimics via intracellular self-assembly is developed. The URG is prepared by enwrapping ALA-loaded polyamide-amine dendrimers in red blood cell membrane vesicles with a further surface modification of G-quadruplex-structured AS1411. The URGs realize "1 O2 -•OH" uninterrupted generation through "recycling waste" in two steps: i) PpIX generates 1 O2 under laser irradiation; and ii) the photoinactive metabolite heme self-assembled with AS1411 to catalyze H2 O2 conversion into •OH. Interestingly, the specific generation of 1 O2 in mitochondria and •OH in nuclei further augments the free-radical-induced damage. It is demonstrated that URG can continuously produce free radicals for 6 h postirradiation, and shows 3.3-times more than that of the nonassembly group, achieving nearly 80% regression of tumors in vivo.

Photochemical Internalization of Etoposide Using Dendrimer Nanospheres Loaded with Etoposide and Protoporphyrin IX on a Glioblastoma Cell Line.

Glioblastoma multiforme (GBM) is the most common malignant primary neoplasm of the adult central nervous system originating from glial cells. The prognosis of those affected by GBM has remained poor despite advances in surgery, chemotherapy, and radiotherapy. Photochemical internalization (PCI) is a release mechanism of endocytosed therapeutics into the cytoplasm, which relies on the membrane disruptive effect of light-activated photosensitizers. In this study, phototherapy by PCI was performed on a human GBM cell-line using the topoisomerase II inhibitor etoposide (Etop) and the photosensitizer protoporphyrin IX (PpIX) loaded in nanospheres (Ns) made from generation-5 polyamidoamine dendrimers (PAMAM(G5)). The resultant formulation, Etop/PpIX-PAMAM(G5) Ns, measured 217.4 ± 2.9 nm in diameter and 40.5 ± 1.3 mV in charge. Confocal microscopy demonstrated PpIX fluorescence within the endo-lysosomal compartment, and an almost twofold increase in cellular uptake compared to free PpIX by flow cytometry. Phototherapy with 3 min and 5 min light illumination resulted in a greater extent of synergism than with co-administered Etop and PpIX; notably, antagonism was observed without light illumination. Mechanistically, significant increases in oxidative stress and apoptosis were observed with Etop/PpIX-PAMAM(G5) Ns upon 5 min of light illumination in comparison to treatment with either of the agents alone. In conclusion, simultaneous delivery and endo-lysosomal co-localization of Etop and PpIX by PAMAM(G5) Ns leads to a synergistic effect by phototherapy; in addition, the finding of antagonism without light illumination can be advantageous in lowering the dark toxicity and improving photo-selectivity.

Investigation of the phenomenon of photobleaching and evaluation of the effectiveness of treatment using a led photodynamic therapy device on optical phantoms

The mortality rate from various oncological diseases remains consistently extremely high, even in spite of recent breakthrough discoveries in the field of cancer therapy [1-3]. One of the most promising cancer treatments is photodynamic therapy (PDT) combined with fluorescence diagnostics (FD) [4, 5]. This study aims to evaluate the efficiency of the PDT procedure using an experimental LED device for photodynamic therapy and fluorescence diagnostics of neoplasms by the level of photobleaching of the photosensitizer protoporphyrin IX (PPIX) in optical phantoms of the skin. The results of spectral measurements showed that the LEDs is able to effectively induce oxidative stress in cancer cells, and due to the presence of a video system, it becomes possible to register the level of tissue fluorescence.

Development of new methods for subcellular diagnostics and therapy of atherosclerosis aimed at eliminating dysfunctional mitochondria.

Background and Aims: Our goal was investigated the relationship of PpIX-induced 5-ALA accumulation on platelet-derived cybrid lines with mutated mitochondria and THP-1 monocytes. There is a photosensitizer protoporphyrin IX (PpIX) widely used in clinical practice, which is produced by 5-aminolevulinic acid (5-ALA) in mitochondria in a cascade of heme synthesis reactions. When 5-ALA is supplied from the outside, the overproduced PpIX cannot be quickly utilized by the available amount of ferrochelatase into the final product - heme and, therefore, accumulates in cells at an increased concentration.

Photodynamic therapy of cutaneous T-cell lymphoma cell lines mediated by 5-aminolevulinic acid and derivatives.

The delta-amino acid 5-aminolevulinic acid (ALA), is the precursor of the endogenous photosensitiser Protoporphyrin IX (PpIX), and is currently approved for Photodynamic Therapy (PDT) of certain superficial cancers. However, ALA-PDT is not very effective in diseases in which T-cells play a significant role. Cutaneous T-cell lymphomas (CTCL) is a group of non-Hodgkin malignant diseases, which includes mycosis fungoides (MF) and Sézary syndrome (SS). In previous work, we have designed new ALA esters synthesised by three-component Passerini reactions, and some of them showed higher performance as compared to ALA. This work aimed to determine the efficacy as pro-photosensitisers of five new ALA esters of 2-hydroxy-N-arylacetamides (1f, 1g, 1h, 1i and 1k) of higher lipophilicity than ALA in Myla cells of MF and HuT-78 cells of SS. We have also tested its effectiveness against ALA and the already marketed ALA methyl ester (Me-ALA) and ALA hexyl ester (He-ALA). Both cell Myla and SS cells were effectively and equally photoinactivated by ALA-PDT. Besides, the concentration of ALA required to induce half the maximal porphyrin synthesis was 209μM for Myla and 169μM for HuT-78 cells. As a criterion of efficacy, we calculated the concentration of the ALA derivatives necessary to induce half the plateau porphyrin values obtained from ALA. These values were achieved at concentrations 4 and 12 times lower compared to ALA, according to the derivative used. For He-ALA, concentrations were 24 to 25 times lower than required for ALA for inducing comparable porphyrin synthesis in both CTCL cells. The light doses for inducing 50% of cell death (LD50) for He-ALA, 1f, 1g, 1h and 1i were around 18 and 25J/cm2 for Myla and HuT-78 cells respectively, after exposure to 0.05mM concentrations of the compounds. On the other hand, the LD50s for the compound 1k were 40 and 57J/cm2 for Myla and HuT-78, respectively. In contrast, 0.05mM of ALA and Me-ALA did not provoke photokilling since the concentration employed was far below the porphyrin saturation point for these compounds. Our results suggest the potential use of ALA derivatives for topical application in PDT treatment of MF and extracorporeal PDT for the depletion of activated T-cells in SS.

Hyaluronic Acid Coated Liposomes Co-Delivery of Natural Cyclic Peptide RA-XII and Mitochondrial Targeted Photosensitizer for Highly Selective Precise Combined Treatment of Colon Cancer

BackgroundNatural cyclopeptide RA-XII, isolated from Rubia yunnanensis, is a promising chemotherapeutic agent for colon cancer. The photosensitizer protoporphyrin-IX attached with triphenylphosphonium (TPP) could possess mitochondria targeting capacity and exert photodynamic therapy (PDT) by inducing oxidizing damage to the mitochondria and cell apoptosis eventually. In this work, pH-sensitive liposomes were constructed to simultaneously deliver RA-XII as a chemotherapeutic drug and modified porphyrin as a mitochondria-targeting photosensitizer to treat colon cancer, and verified its mechanism of action and antitumor therapeutic efficacy.MethodsThe colon cancer targeting liposome nanoparticle RA/TPPP-Lip was synthesized using thin film hydration. The therapeutic effect and targeting ability of RA/TPPP-Lip was investigated in vitro. And use HCT116 cell allogeneic subcutaneous transplantation tumor model to investigate the anti-tumor and targeting effects of RA/TPPP-Lip in vivo.ResultsRA/TPPP-Lip gained the targeting ability through surface-modified HA to increase the accumulation of RA-XII and TPPP in colon cancer cells. A series of in vitro experimental results showed that TPPP produced cytotoxic ROS under laser irradiation to directly damage cell mitochondria and played a combined role with RA-XII, making RA/TPPP-Lip the best colon cancer cell growth inhibitory effect. Furthermore, in vivo antitumor experiments showed that the RA/TPPP-Lip substantially accumulated at the tumor site and efficiently repressed tumor growth in nude mice.ConclusionWe have successfully designed a new cancer-targeted nanomedicine platform (RA/TPPP-Lip) for the collaborative treatment of colon cancer, which can achieve the targeted continuous release of multiple therapeutic drugs. This work provides a new strategy for precise combination therapy, which may promote the further development of collaborative cancer treatment platforms.

The mechanism of 5-aminolevulinic acid photodynamic therapy in promoting endoplasmic reticulum stress in the treatment of HR-HPV-infected HeLa cells.

5-aminoketovaleric acid, as a precursor of the strong photosensitizer protoporphyrin IX (PpIX), mainly enters the mitochondria after entering the cell, and the formed PpIX is also mainly localized in the mitochondria. So at present the research on the mechanism of 5-aminoketovalerate photodynamic therapy (ALA-PDT) mainly focuses on its impact on mitochondria. There are few reports on whether ALA-PAT can affect the endoplasmic reticulum and trigger endoplasmic reticulum stress (ERS). Here we investigated the effects of ALA-PDT on endoplasmic reticulum and its underlying mechanisms in high-risk human papillomavirus (HR-HPV) infection. The human cervical cancer cell line HeLa (containing whole genome of HR-HPV18) was treated with ALAPDT, and cell viability, ROS production, the level of Ca2+ in the cytoplasm and apoptosis were evaluated by CCK8, immunofluorescence and flow cytometry, respectively. The protein expression of the markers of ERS and autophagy and CamKKβ-AMPK pathway was examined by western blot. The results showed that ALA-PDT inhibited cell viability of HeLa cells in vitro; ALA-PDT induced autophagy in HeLa cells ; ALA-PDT induced autophagy via the Ca2+-CamKKβ-AMPK pathway, which could be suppressed by the inhibition of ERS;ALA-PDT induced ERS-specific apoptosis via the activation of caspase 12. Our study demonstrated that ALA-PDT could exert a killing effect by inducing HeLa cell apoptosis, including endoplasmic reticulum-specific apoptosis. Meanwhile, ERS via the Ca2+ -CamKKβ-AMPK pathway promoted the occurrence of autophagy in HeLa cells. Inhibition of autophagy could increase the apoptosis rate of HeLa cells after ALA-PDT, suggesting that autophagy may be one of the mechanisms of PDT resistance; The Ca2+-CamKKβ-AMPK pathway and autophagy may be targets to improve the killing effect of ALA-PDT in treating HR-HPV infection.

Peptide-targeted dendrimeric prodrugs of 5-aminolevulinic acid: A novel approach towards enhanced accumulation of protoporphyrin IX for photodynamic therapy

Photodynamic therapy (PDT) is a promising approach for the targeted treatment of cancer and various other human disorders. An effective, clinically approved approach in PDT involves the administration of 5-aminolevulinic acid (ALA) to generate elevated levels of the natural photosensitiser protoporphyrin IX (PpIX). The development of prodrugs of ALA is of considerable interest as a means to enhance the efficiency and cell selectivity of PpIX accumulation for PDT applications. In this work a novel peptide-targeted dendrimeric prodrug of 5-aminolevulinic acid (ALA) 13 was synthesised which displays nine copies of ALA on a core structure that is linked to a homing peptide for targeted delivery to a specific cancer cell type. The synthesis was accomplished effectively via a flexible, modular solid phase and solution phase route, using a combination of solid phase peptide synthesis and copper-catalysed azide-alkyne cycloaddition chemistry. The prodrug system shows a sustained and enhanced production of protoporphyrin IX (PpIX) in the MDA-MB-231 cell line that over-expresses the epidernal growth factor receptor (EGFR+) in comparison to equimolar ALA and the corresponding non-targeted ALA dendrimer (nine copies of ALA). This study provides a proof of concept for the development of a new generation of prodrugs for ALA-based photodynamic therapy that can deliver an enhanced ALA payload to specific tissue types.

Introduction of a model of skin lesions on rats and testing of dissolving microneedles containing 5-aminolevulinic acid

Topical photodynamic therapy (PDT) is widely used to treat non melanoma skin cancers. It consists of topically applying on the skin lesions a cream containing a prodrug (5-aminolevulinic acid (5-ALA) or methyl aminolevulinate (MAL)) that is then metabolized to the photosensitizer protoporphyrin IX (PpIX). Light irradiation at PpIX excitation wavelength combined with oxygen then lead to a photochemical reaction inducing cell death. Nevertheless, this conventional PDT treatment is currently restricted to superficial skin lesions since the penetration depth of the prodrug is limited and hampers the production of PpIX in deep seated lesions. To overcome this problem, dissolving microneedles (MNs) included in a square flexible patch were developed. This easy-to-handle MN-patch is composed of 5-ALA mixed with hyaluronic acid (HA) and has the ability to dissolve after skin application. To evaluate the efficiency of this MN-patch in vivo, a skin lesion model has been developed on rats by applying UV-B illuminations. After 40 UV-B illuminations, histological and pharmacokinetic controls confirmed that the rats presented skin lesions. Once the rat skin lesion model has been validated, it was demonstrated that the MNs penetrated into the skin and fully dissolved in one hour on most of the rats. After one hour, the fluorescence images showed that the MN-patch produced a consequent and homogeneous level of PpIX. Overall, the dissolving MN-patch is a recent technology that has interesting features and several preclinical investigations should be led to compare its efficiency to that of the conventional treatment for PDT of non melanoma skin cancers.