Date Published: January 20, 2019
Publisher: John Wiley and Sons Inc.
Author(s): Zhi Li, Cuifeng Wang, Huihui Deng, Jiamin Wu, Huan Huang, Ran Sun, Hongbo Zhang, Xiaoxing Xiong, Min Feng.
Advanced melanoma can rarely be cured. Photodynamic therapy (PDT) readily eradicates the primary melanoma but has limited ability to destroy the spreading tumor cells unless supported by other combinative interventions to augment systemic antitumor immunity. Based on the previously synthesized penetration‐enhancing biomaterials, a topically administered nanoformulation is developed, which profoundly assists 5‐aminolevulinic acid (5‐ALA) in circumventing skin barrier to be selectively delivered to tumor cells. After endocytosis, accumulated 5‐ALA is efficiently metabolized to a photosensitizer protoporphyrin IX (PpIX) which stimulates a large production of cytotoxic reactive oxygen species (ROS) under illumination. Accompanied by the robust inflammatory responses followed by primary tumor destruction, CD4+CD8+ double positive T cells are highly boosted to harness host immunity to purge metastases in lymphoid organs. Compared with dacarbazine and programmed death 1 (PD‐1) antibody, this treatment in advanced melanoma murine models, achieves a striking curable rate of 90% without melanoma prognostic markers LDH and S‐100B detection, followed by a relapse‐free survival rate of 83.33% in 300 days. Moreover, the cured mice’s immune system function recovers to an extent similar to healthy mice without prolonged or exaggerated inflammation. This study using the synergistic biomaterials approach may thus render 5‐ALA‐mediated PDT a potentially curative therapy for advanced melanoma in clinic.
Malignant melanoma causes most of skin cancer related deaths, although it accounts for only 4% of skin cancers diagnosed.1 At very early stage, surgical resection can reach more than 90% curable rate; however, it is very difficult to effectively cure the melanoma once distant metastases occur. Hence, various therapeutic strategies including chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations of different types of treatments were employed to treat the advanced‐stage melanoma.2 Conventional surgery and chemotherapy normally elicit immunosuppressive effects that allow cancer cells to evade immune surveillance. Besides of the growing success seen with targeted therapies such as serine/threonine‐protein kinase B‐raf (BRAF) and mitogen‐activated protein kinase kinase (MAP2K, also called MEK) inhibitors,3 effective immunotherapeutic approach has greatly revolutionized the melanoma treatments including blockade of immune‐inhibitory receptors on activated T cells; for example, using monoclonal antibodies against cytotoxic T‐lymphocyte‐associated antigen 4 (CTLA‐4), PD‐1, and programmed death ligand 1 (PD‐L1).4 In terms of survival outcomes, immunotherapy promises to be most significant than any other forms of treatment when tumor metastasis occurs. However, there still exist several obstacles in the field of immunotherapy, such as therapeutic resistance and affordability. In addition to limited success only occurring among few immune responsive melanoma patients, immunotherapy may induce severe autoimmunity against normal tissues.5 High cost will weigh heavily on health‐care systems that are already overburdened, in part by the cost of cancer care. Therefore, it still opens the opportunities to develop an alternative patient‐sparing and economic strategy to treat advanced melanoma.
PDT has great potential to become an ideal oncological intervention for metastatic melanoma which is able to selectively destroy the primary tumor and its microenvironment and at the same time provoke the immune system to attack any remaining or recurring melanoma cells. However, the fact that PDT has yet been accepted as a first‐line clinical option for cancer patients is mainly because of its inefficiency. For example, topically applied 5‐ALA is limited by poor skin penetration and low bioavailability, and only indicated for some nonmalignant skin cancers. Here, we show that 5‐ALA is incorporated into a nanocarrier constituted by CDG2 and HA, as named as CAH. Along with enhanced skin penetration, nanosized CAH facilitates the cellular uptake of 5‐ALA across cell membrane barrier, followed by increased PpIX production. These improvements are partly attributed by skin‐penetrating enhancers in CAH, such as PAMAM‐G2, HA and α‐CD which are previously well‐documented in dermal and transdermal drug delivery.9, 10, 11 Additionally, in contrast to diffusion‐ or transporter‐mediated 5‐ALA cell entry routes, it is more likely that CAH is internalized by melanoma cells via multiple endocytic pathways to increase the cellular accumulation of 5‐ALA, leading to enhanced PpIX formation. Likewise, alterations in internalization pattern also induce intracellular re‐localization of PpIX. In contradiction to some previous reports,27 we find CAH‐induced PpIX is largely localized in the plasma membrane instead of mitochondria where 5‐ALA is initially converted into PpIX. It may be inferred that PpIX is formed in mitochondria after 5‐ALA dissociated from CAH, but rapidly diffuses to plasma membrane due to its lipophilicity. This phenomenon may be explained by varying subcellular redistribution of PpIX depending on different incubation time.28
In summary, our work presents a topically applied PDT nanoformulation (CAH) to cure advanced melanoma in murine models. CAH is capable of overcoming multiple biological barriers for selective delivery of 5‐ALA to melanoma cells. Concurrently with a robust destruction to primary melanoma, CAH‐mediated PDT treatment elicits durable and systemic immunological effects to combat metastases and recurring tumors depending on the activation of CD4+CD8+ double positive T cells. As a result, a striking curative rate of 90% and relapse‐free survival rate of 83% are achieved without immune‐related adverse effects. Thus, such a cost‐effective treatment without adjuvant immunotherapy might have the great potential to be used clinically for advanced melanoma.
Chemicals and Antibodies: N,Nʹ‐carbonyldiimidazole was obtained from Aldrich. α‐Cyclodextrin was purchased from Shandong Zhiyuan Biotechnology (China). PAMAM‐G2 was synthesized as previously described.[[qv: 12b]] HA (M.W. 200–400 kDa, Cosmetic grade) was obtained from Bloomage Freda BioPharm (China). 5‐ALA hydrochloride was purchased from MedChem Express (USA). Pharmaceutical‐ and cosmetic grade cream bases composed of lecithin, Vaseline and glycerol were purchased from Baixiaotang (China). (4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), DMEM medium, fetal bovine serum (FBS), and trypsin were obtained from Gibco (Canada). Cy5 mono NHS ester was obtained from GE Healthcare (UK). LysoTracker Green DND‐26 and MitoTracker Green FM were obtained from Life Technologies (USA). Annexin V‐FITC apoptosis detection kit was purchased from BestBio Biology (China). Fluorometric intracellular ROS kit was purchased from Sigma‐Aldrich (USA). Anti‐mouse PD‐1(CD279) was purchased from Bioxell Life Sciences (USA). APC‐R700 anti‐mouse CD45 antibody, FITC anti‐mouse CD3e antibody, PE anti‐mouse CD8a antibody, APC‐H7 anti‐mouse CD4 antibody, APC anti‐mouse CD44 antibody, and PE‐Cy7 anti‐mouse CD62L antibody were purchased from BD Pharmingen (USA). Mouse anti‐melanoma antibody [HMB45+M2‐7C10+M2‐9E3] was purchased from Abcam (UK). Rabbit anti‐Ki‐67 and rabbit anti‐S‐100B were purchased from Bioss Biology (China). Mouse soluble protein‐100 (S‐100) ELISA kit, mouse interleukin 2 (IL‐2) ELISA kit, mouse interleukin 6 (IL‐6) ELISA kit, mouse tumor necrosis factor (TNF‐α) ELISA kit, and mouse interferon gamma (IFN‐γ) ELISA kit were purchased from Cusabio (USA).
The authors declare no conflict of interest.