This is the first report showing that both stages of Leishmania are intrinsically susceptible to the photolytic activities of soluble and cationic Zn−/Si-Pcs (csPcs) examined ( and [A–B]). Since the axenic amastigotes are closer to the disease-causing stage of Leishmania, their intrinsic and irrevocable susceptibility to csPc-mediated cell death is especially relevant in considering csPcs as agents for therapeutic PT against cutaneous leishmaniasis.
Photolytic activity of the csPcs requires their uptake by cells (), consistent with the outcome of our observations with endogenously generated URO 
. Additions of anilinium or pyridyloxy groups, axial ligands and/or PEGylation to the core structure of the Pc () apparently facilitate the cellular uptake of these csPcs. These modifications increase their cationicity for enhancing interaction with the negatively charged cell surface, and their solubility for increased bioavailability 
and decreased self-quenching 
. Subcellular localization of the representative csPcs () suggests that the mechanisms of their cellular uptake follow at least 2 different pathways, common to both Leishmania
and macrophages: endocytosis for pyridyloxy csPcs, e. g. 14/15, and plasma and mitochondrial membrane transport of di-anilinium csPcs, i. e. Pc 3.5. It is not known whether the mitochondrial import of this csPc utilizes a specific transporter, as reported for a different Pc series, e. g. Si-Pc4 
. Further study of the structure-function relationships of these and other csPcs are needed to elucidate the precise mechanisms of their cellular uptake and trafficking.
Our results together with those from previous work show that the subcellular targeting differences of the PS figure significantly in the photolytic phenotype observed. The subcellular targeting specificity of the effective csPcs presented here differs from that, which we reported previously, for endogenously induced URO 
and exogenously applied AlPhCl 
. The csPcs accumulate gradually in Leishmania
, akin in timeframe to the neogenesis of URO in porphyric mutants 
, but in different sites, resulting in the manifestation of very different phototoxic phenotypes. Flagellar motility was rapidly paralyzed by light exposure of the uroporphyric mutants when URO began to emerge in their cytosol 
, but not when Leishmania
was pre-loaded with csPcs in their endosome/phagolysosomes or mitochondria. These PS-sensitized Leishmania
do not lose their viability immediately after illumination in sharp contrast to the outcome of those treated with membrane-associated AlPhCl 
. The cellular targeting specificity of these and other csPcs warrants further study to understand their mechanisms in relation to their observed differences in photodynamic properties.
In the present study, evidence is also presented for the first time that the endocytic PS, like csPcs 14/15, are potentially useful for therapeutic PT against phagolysosomal pathogens, e. g. Leishmania
spp. The specificity of these PS for targeting phagolysosomal Leishmania
accounts more for their effectiveness than their intrinsic photolytic activities, as the mitochondrial csPcs are more photolytic to promastigotes, but less leishmanolytic against those in infected cells than the endocytic csPcs 14/15 (). The endocytic csPcs are expected to be effective for PT in vivo
by just clearing the infection of some infected MCs so that they, once free from Leishmania
-mediated immunosuppression, are able to initiate effective immunity to clear the remaining infection. This scenario is consistent with some measure of success of PT using other PS reported against clinical cutaneous leishmaniasis 
. The use of endocytic csPcs is expected to significantly enhance both pharmacological effectiveness of PT as well as the post-therapeutic immune clearance of Leishmania
infection. For such applications, csPcs may be further modified for lysosomal activation 
to increase the margin of parasite versus host selectivity.
Our in vitro
data presented support our proposal that the PS-loaded Leishmania
are potentially useful carriers to deliver drugs/vaccines to the appropriate site for pharmacological/immunological activation 
pre-loaded with csPcs provide an additional carrier inducible for destruction () as alternatives to the uroporphyrinogenic mutants 
. The csPcs appear “locked up” in the cell organelles more tightly than membrane-associated AlPhCl 
, thereby avoiding “leaching out” to sensitize host cells for photolysis, as found with the latter. Pre-illumination of these csPc-loaded Leishmania
eliminates their ability to grow, thereby increasing the safety margin of their future applications (). Also, the clearance of Leishmania
from infected cells requires no additional illumination, thereby simplifying the experimental steps. While persistence of a few Leishmania
below detection can never be ruled out, they are expected to succumb to post-PT immune clearance under in vivo
conditions, as noted previously 
Evidence is further provided for the first time that specific antigen can be expressed by Leishmania for photolytic delivery after PS-loading to DC or BDMC to elicit a T cell response, supporting our proposal for their utility as a vaccine carrier in immuno-prophylaxis and –therapy. Transfection of Leishmania to express OVA makes it possible to photolytically deliver it as a surrogate vaccine for in vitro evaluation of T cell specific immune response (). Significantly, csPc-loaded transfectants are able to deliver OVA to DCs and MCs for appropriate processing. Pre-illumination of csPc 14/15-loaded transfectants gave the most consistent results, suggesting that the photolytic environment of the PT preserve not only the carrier capacity of the transfectants but also the antigenicty of OVA epitopes in these cells. Delivery of OVA by photo-inactivated Leishmania to BDMC for this activity is especially impressive, as it is higher even than that produced by the lysates of these Leishmania that were supplied to APC in equivalent amounts (). While DCs and MCs are susceptible to the infection by the csPc-loaded transfectants and illumination of these infected cells cleared the infection (), delivery of OVA in this way for antigen presentation produced less consistent results (not shown). Work is still on-going to optimize the experimental conditions. OVA SIINFEKL-MHC Class I co-presented by the infected DCs and BDMCs is functionally active, since such APCs are capable of activating SIINFEKL-specific CD8+ T cells. Work is underway to evaluate the lysosomal delivery of OVA for presentation of different OVA epitopes to specific CD4+ T cells. Completion of the work with this and other defined antigens is expected to provide the necessary foundation for future evaluation of vaccine candidates photolytically delivered by Leishmania against other diseases.