Light induced therapeutic modalities, because of minimal invasiveness and high spatiotemporal selectivity, are attracting attention of medical and research community for management of various chronic ailments such as cancer and chronic wounds. These therapeutic effects can be induced by activation of either an endogenous or exogenously applied photoaceeptor (Figure 1). At LBAD, studies are being carried out to explore the possible applications of various light based therapeutics in disease management.
Figure 1 : Possible ways of generating light induced therapeutic effects.
(1) Photodynamic therapy (PDT) for tumor
Amongst the different approaches, photodynamic therapy (PDT) is a clinically approved modality for the treatment of cancer and macular degeneration. Currently, this modality is also being explored for the treatment of other diseases such as superficial bacterial infections and restenosis of arteries.
PDT utilizes light and oxygen in combination with a photosensitizer (PS) to achieve cellular destruction through production of cytotoxic reactive oxygen species, resulting in direct cancer cell death and immune cell activation mediated tumor regression. Although the promise generated by PDT is far-reaching, it still suffers from limitation such as poor solubility of most of the PS in aqueous environment and suboptimal targeting of tumor areas with poor vasculature.
(i) Biomolecule conjugation for improved PS delivery and PDT outcome : Our previous studies on Chlorin p6, (Cp6) a chlorophyll derivative prepared in-house had shown promising results in Cp6 accumulation preferentially in cancer cells and pH dependent variation in cell death mode after PDT. In hamster cheek pouch model, Cp6 given intraperitoneally (1.5 mg/kg body weight) showed good tumor selectivity in case of tumors smaller than 5 mm, led to complete tumor damage subsequent to PDT and rapid excretion from normal tissue. For larger tumor sizes accumulation of the drug was poor for systemically administered drug. Later studies have shown that the delivery and photodynamic efficacy of Cp6 in larger tumors improved upon conjugation with histamine. Histamine conjugated Cp6 (Cp6-his) showed ~10 times higher uptake in oral cancer cells, enhancement in phototoxicity, improvement in tumor selectively and complete regression of larger tumor of volumes up to 1000 mm3.
(ii) PS metal complex for multimodal PDT: For further developing the use of Cp6 as a multimodal agent for PDT and photon activation therapy, an iodinated–Chlorin p6 copper complex (ICp6–Cu) was prepared. Results show that compared to the Cp6, ICp6-Cu is more effective for PDT of cancer cells under hypoxic condition. Further, pre-treatment of cells with 20 μM and 30 μM ICp6-Cu for 3 h was found to enhance the X-ray-induced cytotoxicity in vitro with sensitization enhancement ratios of 1.8 and 2.8, respectively.
(iii) Nanoparticles assisted PS delivery and targeting for anticancer PDT: Another promising approach to improve PDT efficacy is nanoparticle (NP) based PS delivery. This approach is receiving attention because NP can be used to encapsulate poorly soluble drugs, to improve drug stability, modify their blood circulation and also reduce side effects of drugs. For instance, organically modified silica nanoparticle (ORMOSIL) and curcumin complex conjugated with hyaluronic acid (Figure 2) resulted in improved stability, uptake and PDT efficacy of curcumin in cancer cell lines and spheroids.
Figure 2 : Schematic diagram showing SiNp preparation and curcumin encapsulation, for anticancer application
(iv) Ligand nanoparticle conjugation for improved anticancer PDT outcome: Similarly, the use of ORMOSIL as a delivery vehicle (figure 3) also led to improved stability, uptake and PDT efficacy of another hydrophobic drug; pheophorbide (PPa). The PPa delivery to cancer cells was further improved by conjugating a with ligand folic acid.
Figure 3 : Schematic depiction of synthesis of plain and folic acid conjugated ORMOSIL entrapping PPa, for anticancer application.
(2) Antimicrobial Photodynamic Therapy (APDT) for antibiotic resistant microbes
Antibiotic resistance in now a global menace. In wound care practice, this has been attributed as one of the leading causes of lower limb amputations, morbidity and mortality. Antimicrobial photodynamic therapy (APDT), because of the selectivity and remote possibility of resistance generation, is a promising alternative to antibiotics.
The other advantages of APDT are inactivation of virulence factors, proteases enzymes, and modulation of host inflammation response. Thus APDT is a suitable methodology for bacteria inactivation and chronic wound management. Our earlier in vitro studies on the antimicrobial effects of APDT showed that photosensitization with phenothiazinium dyes induced not only lethal effect but also some favorable responses at sub lethal doses e.g. decrease in virulence of Pseudomonas aeruginosa [10]. Photodynamic treatment was also found to be effective for inactivation of antibiotic resistant Staphylococcus biofilms. Since the interaction of photosensitizer with the cell envelop plays a critical role in photoinactivation of bacteria, studies on cell surface alterations induced by toludine blue (TBO) mediated photodynamic treatment on a Gram-positive bacterium, Staphylococcus aureus and a Gram-negative bacterium, Escherichia coli were carried out using Atomic force microscopy.
(i) Polypeptide conjugation for improved APDT outcome : One of the main concerns in the use of APDT is the inability of anionic photosensitizers to cross the complex cell envelop of Gram negative bacteria. To overcome such problem, PS conjugated to cationic molecules such as polylysin showed considerable promise. Our studies showed that topical antimicrobial photodynamic treatment (APDT) mediated by poly- L-lysin conjugated chlorin p6 (pl-cp6) not only reduces the bacterial load , but also lowers the hyper inflammatory response caused by bacteria infection. Further, pl-cp6 mediated APDT expedites healing in both methicillin resistant S. aureus (MRSA) and P. aeruginosa infected wounds of mice by attenuating degradation of collagen, enhancing epithelialization, hydroxyproline content and collagen remodeling.
(ii) Light fluence optimization for improved APDT outcome: In MRSA infected wounds of diabetic mice, our results demonstrate that compared to single APDT at high light fluence, multiple APDT at lower fluence shows better healing response in MRSA infected wounds in diabetic mice. This has been shown to be due to reduced damage to host cells and enhanced level of antioxidants as well as angiogenic growth factors. Therefore, a suitable low fluence APDT window can be optimized that results not only in significant bacteria inactivation, negligible damage to the inflammatory cells, but also concomitant increase in cell proliferation response of wounds.
(iii) Development of nanoformulations and dressings for improved APDT and wound healing outcome: Some of the challenges in APDT in-vivo is rapid degradation of hydrophobic drugs under aqueous environment and uncontrolled PS release at wound site. Our works have also shown that conjugation with hyaluronic acid improves the stability, photobactericidal action and wound healing efficacy of the hydrophobic drug; Curcumin.
Figure 4 : Schematic of preparation of curcumin HA conjugate for wound healing application
Further, to achieve a sustained PS release in wounds, we are employing different approaches such as development of suitable dressings and hydrogels. A collagen based wound dressing containing the HA-cur nanoformulation has been prepared. This dressing has shown good promise for both bacteria inactivation and improved wound closure outcome in diabetic mice (Figure 5).
Figure 5 : Schematic of preparation of collagen biofilm with curcumin HA conjugate for wound healing application
(iv) Nanoparticles assisted photothermal antibacterial therapy: We are further investigating the applicability of metal nanoparticles, specifically gold nanorods, mediated photothermal treatment as an approach for management of anti-biotic resistant bacterial infections. Nanoparticles based approaches based photothermal treatment has potential as an alternative approach for anti-bacterial applications. Herein, metal nanoparticles are used as transducers to convert the energy from photon to thermal energy mediated by phonon vibrations. The approach has potential to be applied as an adjunct therapy for cancer, where it may assist in producing more desirable results with minimum collateral damages to surrounding healthy cells. In our study we found that the photothermal treatment mediated by polyelectrolyte-coated gold nanaorods resulted in 90% cytotoxicty in bacteria (Figure 6).
Figure 6 : Schematic representation of photothermal induced anti-bacterial effect of ployelectrolyte coated gold nanorods (PSS-GNRs and PDDAC-GNRs)
(v) Optical spectroscopy for monitoring APDT outcome: Use of optical spectroscopic and imaging techniques to monitor efficacy of therapeutics meant for improved wound healing is another area with potential for medical application. Earlier, in one of our studies, to explore the efficacy of APDT for control of wound infection, polarization sensitive optical coherence tomography (PSOCT) was used by us. While use of PSOCT enables only morphological assessment, integration of OCT with Raman spectroscopy allows a complete morpho-biochemical mapping of the wounds subjected to APDT or other therapuetic interventions and is being pursued.
(vi) Optical imaging for assessment bacteria load in wounds: Bacteria load is one of the crucial factors that delay wound healing. Currently, the established clinical practices for wound infection diagnosis rely on subjective, qualitative visual examination under room lighting/white light for clinical signs and symptoms, with confirmation via time taking processes such as quantitative histology and microbiological assays. Hence, timely detection of bacterial could dispel diagnostic uncertainty and allow evidence based assessment of antibacterial therapy. Our recent works involve development of diagnostic approach for non-invasive detection of bacterial load in wounds. The method makes use of 5-amino laevulinic acid induced rapid biosynthesis of protoporphyrin IX by bacteria and detection of the red fluorescence in wounds upon blue light excitation.
(3) Use of low level light therapy for burns and chronic wounds:
Excitation of endogenous chromophores such as cytochromes by visible light leads to increase in metabolic rate, proliferation capability, growth factor secretion in cells. This therapeutic intervention, named low level light therapy (LLLT) or photobiomodulation has shown good promise for management of burns, chronic wounds.
Use of LLLT for management of burn injury: For instance, a pre clinical study shows that single exposure near infrared (830 nm) at a fluence of 3 J/cm2 enhanced wound healing in full thickness burns induced in Swiss albino mice.
Use of LLLT for management of diabetic foot ulcers: clinical study suggests that LLLT (660 ± 20 nm, ~3 J/cm2), used as an adjuvant to conventional antibiotics treatment options can improve rate of healing in diabetic foot ulcers (Figure 7).The results showed that after two weeks, the percentage ulcer area reduction in the LLLT group, as compared to the control group was considerably higher.
Figure 7 : Effect of LLLT (660 ± 20 nm, ~3 J/cm2) on DFUs. Wound contraction with respect to day 0, in control and LLLT group
(4) Optogenetics
In the pursuit of exploiting novel technologies for management of metabolic diseases, like diabetes, we are venturing into utilizing optogenetic tools for influencing cellular behavior using light.
Our current focus is photo-induced secretion of hormones.
Figure 8 : Schematic representation of the optogenetics approach for light-induced hormonal release from cells
