Evaluation of bacteriochlorophyll-reconstituted low-density lipoprotein nanoparticles for photodynamic therapy efficacy in vivo

Abstract

Aim: To evaluate the novel nanoparticle reconstituted bacteriochlorin e6 bisoleate low-density lipoprotein (r-Bchl-BOA-LDL) for its efficacy as a photodynamic therapy agent delivery system in xenografts of human hepatoblastoma G2 (HepG2) tumors.

Materials & methods: Bchl-BOA was encapsulated in the nanoparticle low-density lipoprotein (LDL), a native particle whose receptor's overexpression is a cancer signature for a number of neoplasms. Evaluation of r-Bchl-BOA-LDL as a potential photosensitizer was performed using a tumor response and foot response assay.

Results & discussion: When compared with controls, tumor regrowth was significantly delayed at injected murine doses of 2 µmole/kg r-Bchl-BOA-LDL after illumination at fluences of 125, 150 or 175 J/cm(2). Foot response assays showed that although normal tissue toxicity accompanied the higher fluences it was significantly reduced at the lowest fluence tested.

Conclusion: This research demonstrates that r-Bchl-BOA-LDL is an effective photosensitizer and a promising candidate for further investigation.

Figure 1. The lipoprotein-based nanoplatform concept for targeted drug delivery

Reconstituted low-density lipoprotein core labeled with Bchl-BOA is depicted along with the solitary ApoB-100 protein and phospholipid monolayer.

Figure 2. Synthesis of bacteriochlorophyll analogs suitable for low-density lipoprotein reconstitution

(A) Bchl-acid. (B) Bchl-Meester. (C) Bchl-BOC. (D) Bchl-NCS. (E) Bchl-CE. (F) Bchl-2 BOC. (G) Bchl-BOA. (i) CH₂N₂, dichloromethane, room temperature (rt) 1 h, 85%. (ii) N-Boc-1,3-diaminopropane, CHCl₃, reflux/Ar, 48 h, 70%. (iii) Trifluoroacetic acid (TFA), rt/Ar, 1 h, 86%. (iv) 1,1’-thiocarbonyldiimidazole, dichloromethane, reflux/Ar, 4 h, 75%. (v) 5-androsten-17β-amino-3β-yl oleate, dichloromethane, diisopropylethylamine, rt/Ar, 24 h, 86%. (vi) N-Boc-1,3-diaminopropane, 4-N,N-dimethylaminopyridine, dicyclohexylcarbodiimide, dichloromethane/Ar, 40 h, 72%. (vii) TFA, rt/Ar, 1 h. viii) oleoyl chloride, dichloromethane, rt/Ar, 1 h, 63% (vii, viii).

Figure 3. HepG₂-tumored mice pre- and post-photodynamic therapy

(A) Nude mouse bearing a tumor on its left flank. (B) 24 h following photodynamic therapy (PDT; 150 J/cm², 2 µmole/kg Bchl-BOA-LDL), characteristic burn/edema was visible over tumor while (C) scab formation was observed 9 days post-PDT. (D) Complete tumor ablation (arrow) was seen at 60 days post-PDT.

Figure 4. Survival curves for photodynamic therapy-treated and control groups

PDT conditions evaluated are 125, 150 or 175 J/cm² and 2 µmole/kg r-Bchl-BOA-LDL. Survival for untreated, light only (175 J/cm²) and drug alone (2 µmole/kg r-Bchl-BOA-LDL) controls is also displayed. PDT treatments were significantly more effective in controlling tumor regrowth compared with control groups. *p < 0.0001. For all groups, 3 ≤ N ≤ 8. PDT: Photodynamic therapy.

Figure 5. Redox ratios for drug control and untreated mice

Redox ratios of HepG₂ tumor tissue for (A) drug control and (B) untreated mice. Redox ratios of liver tissue for (C) drug control and (D) untreated mice. Redox ratios of muscle tissue for (E) drug control and (F) untreated mice. Redox ratios of kidney tissue for (G) drug control and (H) untreated mice. Fp: Flavoprotein.

Figure 6. Foot response assay

Average maximum morphology scores are shown for photodynamic therapy (PDT) at 125 and 150 J/cm² (n = 3). PDT at 125 J/cm² displayed a significantly lower average maximum morphology score compared with PDT at 175 J/cm². *p < 0.05. The light control (175 J/cm²) demonstrated no morphological change.