Malcolm E. Kenney

Phthalocyanine photosensitizers for photodynamic therapy

Six new aluminum and silicon phthalocyanines have been synthesized and their photocytotoxicity toward V79 cells has been studied. The compounds that have been prepared are: AIPcOSi(CH3)2(CH2),N(CH3)2, I; AIPcOSi(CH3)2(CH2)3N(CH3)3+I−, II; CH3SiPcOSi(CH3)2(CH2)3N(CH3)2, III; HOSiPcOSi(CH3)2(CH2)3N(CH3)2, IV; HOSiPcOSi(CH3)2(CH2)3)3(CH3)3+I−, V; and SiPc[OSi(CH3)2(CH2)3N(CH3)3+I−]2, VI. Relative growth delay values for compounds I‐VI and relative cytotoxicity values for compounds I, II, IV, V and VI have been determined. Compounds I and II have been shown to be comparable in photocytotoxicity to what is presumed to be AIPcOH.xH2O, and compound IV has been shown to have greater activity. The classes of compounds to which these six compounds belong appear to have potential for photodynamic therapy.

Deep Penetration of a PDT Drug into Tumors by Noncovalent Drug-Gold Nanoparticle Conjugates

Efficient drug delivery to tumors is of ever-increasing importance. Single-visit diagnosis and treatment sessions are the goal of future theranostics. In this work, a noncovalent PDT cancer drug-gold nanoparticle (Au NP) conjugate system performed a rapid drug release and deep penetration of the drug into tumors within hours. The drug delivery mechanism of the PDT drug through Au NPs into tumors by passive accumulation was investigated via fluorescence imaging, elemental analysis, and histological staining. The pharmacokinetics of the conjugates over a 7-day test period showed rapid drug excretion, as monitored via the fluorescence of the drug in urine. Moreover, the biodistribution of Au NPs in this study period indicated clearance of the NPs from the mice. This study suggests that noncovalent delivery via Au NPs provides an attractive approach for cancer drugs to penetrate deep into the center of tumors.

Delivery and efficacy of a cancer drug as a function of the bond to the gold nanoparticle surface.

In this feature article, gold nanoparticle conjugates loaded with phthalocyanine-based PDT drugs are prepared and tested for delivery efficiency and PDT efficacy on HeLa cancer cells. It could be shown that the delivery and PDT outcome are strongly affected by the bond that links the drug load to the nanoparticle surface. Whereas labile amino adsorption to the Au nanoparticle surface allows for efficient drug release into the cancer cells and for efficient PDT, a covalent thiol bond to the Au nanoparticle leads to the delivery of the drug into cell vesicles, and no PDT effect is observed. This work highlights the importance of carefully choosing the interaction between drug molecules and the nanoparticle surface.

The synthesis, photophysical and photobiological properties and in vitro structure-activity relationships of a set of silicon phthalocyanine PDT photosensitizers

Jin He1

Delivery of the photosensitizer Pc 4 in PEG–PCL micelles for in vitro PDT studies

The silicon phthalocyanine Pc 4 is a second-generation photosensitizer that has several properties superior to other photosensitizers currently approved by the FDA, and it has shown significant promise for photodynamic therapy (PDT) in several cancer cells in vitro and model tumor systems in vivo. However, because of the high hydrophobicity of Pc 4, its formulation for in vivo delivery and favorable biodistribution become challenging. To this end, we are studying encapsulation and delivery of Pc 4 in block copolymer micelles. Here, we report the development of biocompatible PEG-PCL micelle nanoparticles, encapsulation of Pc 4 within the micelle core by hydrophobic association with the PCL block, and in vitro PDT studies of the micelle-formulated Pc 4 in MCF-7c3 human breast cancer cells. Our studies demonstrate efficient encapsulation of Pc 4 in the micelles, intracellular uptake of the micelle-formulated Pc 4 in cells, and significant cytotoxic effect of the formulation upon photoirradiation. Quantitative estimation of the extent of Pc 4 loading in the micelles and the photocytotoxicity of the micelle-incorporated Pc 4 demonstrate the promise of our approach to develop a biocompatible nanomedicine platform for tumor-targeted delivery of Pc 4 for site-selective PDT.

New phthalocyanines for photodynamic virus inactivation in red blood cell concentrates.

Abstract Cationic phthalocyanines with either aluminum or silicon as the central metal were evaluated for their ability to inactivate viruses in red blood cell concentrates (RBCC) photodynamically. In addition, the virucidal potential of a substituted anionic phthalocyanine, aluminum dibenzodisulfophthalocyanine hydroxide (AlN2SB2POH) was evaluated and compared with that of the much studied anionic aluminum tetrasulfophthalocyanine hydroxide (AIPcS4OH). Based on the rate of inactivation of the lipid‐enveloped vesicular stomatitisvirus (VSV), the viruci dal potential of these phthalocyanines was: HOSiPCOSi(CH3)2(CH2)3N+(CH3)3I‐ (Pc 5) = SiPC[OSi(CH3)2‐(CH2)3N+(CH3)3I‐]2 (Pc 6) > AIPCOSi(CH,)2(CH2)˜N+(CH3)2(CH˜)11CH3I‐ (Pc 21) = A1N2SB2POH = AlPcS4 > HOSiPc[OSi(CH3)2(CH2)3N+(CH3)2(CH2)11CH31–]2(Pc 14) > AIPcOSi(CH3)2(CH2)3N+(CHS)3I‐ (Pc 2). Phthalocy anine ligand 14 and Pc 21 are new phthalocyanines, made by quaternizing known amino analogues. Compared to VSV, the rate of inactivation of Sindbis virus (another model lipid‐enveloped virus) was identical when treated in red blood cells (RBC) with Pc 5 and slightly higher when treated with Pc 6 and AlPcS4OH. Treatment of RBCC containing cell‐free human immunodeficiency virus (HIV‐1) with Pc 5 or AlPcS4OH required 15 min of irradiation to inactivate (>5 log10 reduction) the virus. The extent of HIV‐1 inactivation with AlN2SB2POH was 3.7 log10 after 60 min of red light exposure. The RBC integrity after photosensitization was measured by the ability of the cells to bind to plates coated with poly‐L‐lysine, (which reflects the retention of the RBC surface negative charges) and hemolysis of the cells over a 7 day storage period. The RBC damage using these criteria was most pronounced with Pc 5 and Pc 6 but could be reduced when treatment was in plasma instead of buffer. These data indicate that lipid‐enveloped viruses differ in their sensitivity to phthalocyanine photosensitization. Therefore, for virus sterilization of RBCC for transfusion the ability to inactivate human pathogenic viruses completely will have to be evaluated for each virus. The cationic Pc 5 appears to be a potentially useful virucidal agent.

Phthalocyanine 4 (Pc 4) Photodynamic Therapy of Human OVCAR‐3 Tumor Xenografts

Abstract— Photodynamic therapy (PDT) is a cancer treatment modality utilizing a photosensitizer, light and oxygen. Photodynamic therapy with Photofrin® has been approved by the US. Food and Drug Administration for treatment of advanced esophageal and early lung cancer. Because of certain drawbacks associated with the use of Photofrin, there is a need to identify new photosensitizers for human use. The photosensitizer Pc 4 (HOSiPc‐OSi[CH3]2[CH2]3N[CH3]2) has yielded promising PDT effects in many in vitro and in vivo systems. The aim of this study was to assess the usefulness of Pc 4 as a PDT photosensitizer for a human tumor grown as a xenograft in athymic nude mice. The ovarian epithelial carcinoma (OVCAR‐3) was heterotransplanted subcutaneously in athymic nude mice. Sixty mice bearing OVCAR‐3 tumors (∼80–130 mm3) were divided into six groups of 10 animals each, three for controls and three for treatment. The Pc 4 was given by tail vein injection, and 48 h later a 1 cm area encompassing the tumor was irradiated with light from a diode laser coupled to a fiberoptic terminating in a microlens (Λ= 672 nm, 150 J/cm2,150 mW/cm2). Tumors of control animals receiving no treatment, light alone or Pc 4 alone continued to grow. Of animals receiving 0.4 mg/kg Pc 4 and light, one (10%) had a complete response and was cured (no regrowth up to 90 days post‐PDT), while all others (90%) had a partial response and were delayed in regrowth. Of animals receiving 0.6 mg/kg Pc 4 and light, eight (80%) had a complete response, and two of these were cured. Of animals receiving 1.0 mg/kg Pc 4 and light, six (60%) had a complete response, and two of these were cured. In additional experiments, tumors from animals treated with Pc 4 (1 mg/kg) and light were removed 15, 30, 60 and 180 min post‐PDT, and from these tumors DNA and protein were extracted. Agarose gel electrophoresis revealed the presence of apoptotic DNA fragmentation as early as 15 min post‐PDT. Western blotting showed the cleavage of the 116 kDa native poly (ADP‐ribose) polymerase (PARP) into fragments of ∼90 kDa, another indication of apoptosis, and the presence of p21/WAFl/CIPl (p21) in all PDT‐treated tumors. These changes did not occur in control tumors. Pc 4 appears to be an effective photosensitizer for PDT of human tumors grown as xenografts in nude mice. Early apoptosis, as revealed by PARP cleavage, DNA fragmentation and p21 overexpression, may be responsible for the excellent Pc 4‐PDT response. Clinical trials of Pc 4‐PDT are warranted.

Addressing Brain Tumors with Targeted Gold Nanoparticles: A New Gold Standard for Hydrophobic Drug Delivery?

EGF-modified Au NP-Pc 4 conjugates showed 10-fold improved selectivity to the brain tumor compared to untargeted conjugates. The hydrophobic photodynamic therapy drug Pc 4 can be delivered efficiently into glioma brain tumors by EGF peptide-targeted Au NPs. Compared to the untargeted conjugates, EGF-Au NP-Pc 4 conjugates showed 10-fold improved selectivity to the brain tumor. This delivery system holds promise for future delivery of a wider range of hydrophobic therapeutic drugs for the treatment of hard-to-reach cancers.

Transferrin receptor-targeted theranostic gold nanoparticles for photosensitizer delivery in brain tumors

Therapeutic drug delivery across the blood-brain barrier (BBB) is not only inefficient, but also nonspecific to brain stroma. These are major limitations in the effective treatment of brain cancer. Transferrin peptide (Tfpep) targeted gold nanoparticles (Tfpep-Au NPs) loaded with the photodynamic pro-drug, Pc 4, have been designed and compared with untargeted Au NPs for delivery of the photosensitizer to brain cancer cell lines. In vitro studies of human glioma cancer lines (LN229 and U87) overexpressing the transferrin receptor (TfR) show a significant increase in cellular uptake for targeted conjugates as compared to untargeted particles. Pc 4 delivered from Tfpep-Au NPs clusters within vesicles after targeting with the Tfpep. Pc 4 continues to accumulate over a 4 hour period. Our work suggests that TfR-targeted Au NPs may have important therapeutic implications for delivering brain tumor therapies and/or providing a platform for noninvasive imaging.

Structural factors and mechanisms underlying the improved photodynamic cell killing with silicon phthalocyanine photosensitizers directed to lysosomes versus mitochondria.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4‐loaded cells, membrane components, especially Bcl‐2, are photodamaged and apoptosis, as indicated by activation of caspase‐3 and cleavage of poly(ADP‐ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three‐fold higher affinity constants for liposomes. The hydroxyl‐bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4‐PDT photodamages Bcl‐2 and Bcl‐xL, Pc 181‐PDT causes much less photodamage to Bcl‐2 over the same dose–response range relative to cell killing, with earlier cleavage of Bid and slower caspase‐3‐dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl‐bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.