In Light of Recent Developments,Application of Fluorescence Spectral Analysis in Tumor Diagnosis

Abstract

This paper reviews the background to cancer tumors. Fluorescence spectral analysis has been quantitatively applied to the detection of protoporphyrin IX (Pp‐IX) in transplanted squamous cell carcinoma (SCC) tissue and intra‐operative brain astrocytoma (glioblastoma) tissue after the administration of 5‐aminolevulinic acid (5‐ALA). Coincidence with the emission spectra of Pp‐IX incorporated into the tumor tissues (cryo‐sections) and a standard Pp‐IX in the miceller solution was confirmed. A calibration curve of the fluorescence intensity of Pp‐IX against a known concentration standard of Pp‐IX was established. From the fluorescence detection method (calibration curve), it was found that the longer alkyl‐chain length (methyl‐ and hexyl‐) 5‐ALA induced Pp‐IX in the SCC tissue. Furthermore, an ultrasound treatment enhanced the uptake of Pp‐IX during the topical administration of 5‐ALA derivatives. In the case of intra‐operative observation for the fluorescence intensity of Pp‐IX in the brain astrocytoma tissue, this quantitative detection in the cryo‐section demonstrated the intra‐operative observation for the fluorescence intensities (?, +/?, +, ++, +++) diagnosis was correlated closely with the grades of astrocytoma. The intensity increased exponentially with the cancer grades. In future, fluorescence spectral diagnosis of Pp‐IX in the tumor will be very useful for other clinical diagnosis of tumors using 5‐ALA.     

The photosensitizing effect of the photoproduct of protoporphyrin IX

The photodynamic effect of a photoproduct of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) was investigated in WiDr cells, a human adenocarcinoma cell line. The fluorescence excitation and emission spectra of PpIX and the photoproduct were measured. After 1, 3 or 5 min exposure of the ALA-incubated cells to 140 mW/cm² light at 635 nm, the photoproduct — the chlorin photoprotoporphyrin (Ppp), had an emission band around 670 nm. The Ppp excitation peak at 670 nm is well separated from the PpIX peak at 635 nm. The outcome of photodynamic therapy (PDT) was determined by measuring intracellular fluorescence intensity of propidium iodide (PI) 2 h following PDT and methylene blue (MB) staining 24 h following PDT. A significant increase in the fluorescence intensity of PI was noted when the ALA-loaded cells were exposed to 670 nm light after exposure to 635 nm, indicating enhanced cell membrane inactivation induced by the photodynamic action of the photoproduct. However, the fraction of the cells that survived following the same treatment as measured by MB staining was not significantly affected based on an analysis of variance. The fluorescence of PpIX decayed significantly during 635 nm light exposure. Exposure to light at 670 nm does not lead to any photodegradation of PpIX. The fluorescence of Ppp was bleached during 670 nm light exposure. Exposure of Ppp at 670 nm gives no PpIX back. Thus, the phototransformation of PpIX to Ppp is probably not a reversible process.     

Sonodynamically induced anti-tumor effect with protoporphyrin IX on hepatoma-22 solid tumor

Objective

The purpose of this study was to evaluate sonodynamically induced anti-tumor effect of protoporphyrin IX (PPIX) in mice bearing hepatoma-22 (H-22) solid tumors, and the possible in vivo cell damage mechanism was also investigated.

Methods

The pharmacokinetics of PPIX was analyzed in plasma, skin, muscle and tumor of H-22 bearing mice. Tumors were irradiated with ultrasound (1.43 MHz, I_SATA 3 W/cm², 3 min) for three times at 8, 12 and 24 h after 5.0 mg/kg PPIX administration, respectively. The anti-tumor effects of sonodynamic therapy (SDT) were estimated by the tumor inhibition ratio (volume and weight). The bio-effects of SDT were evaluated by hematoxylin and eosin (H&E) staining, transmission electron microscope (TEM) observation, lipid peroxidation (LPO) measurement and anti-oxidative enzymes (glutathione peroxidase (GSH-PX), catalase (CAT) and superoxide dismutase (SOD)) assay.

Results

A significant anti-tumor effect was obtained by PPIX-mediated sonodynamic therapy (PPIX-SDT). At the fifteenth day after PPIX-SDT, the tumor growth and tumor weight inhibition ratios were 53.84% and 45.86%, respectively. In addition, the structure of tumor tissues and the anti-oxidative enzymes were obviously destroyed after SDT treatment.

Conclusions

A biochemical mechanism was involved in PPIX-SDT in vivo, and the free radicals produced by the synergistic treatment destroying the anti-oxidative system of tumor cells in vivo may play an important role in this action. Also, the thermal effect could not be excluded in inducing damage of cellular structures, like membrane disruption and chromatin condensation under current evaluation in this paper.     

A DFT study on the vibrational spectroscopy of protoporphyrin IX

Infrared and Raman spectra of protoporphyrin IX were recorded. DFT quantum chemical calculations were performed. Optimised molecular geometry, electric charge distribution, vibrational force constants were computed. The normal coordinate analysis and the scaling of the force constants yielded all the necessary data for the simulation of the infrared and Raman spectra and the potential energy distribution calculations. The result was the interpretation of all vibrational modes of the molecule. Conclusions were drawn from the difficulties arisen during the assignment of the vibrational spectra of such large molecules.     

Magnetism of ferriprotoporphyrin IX monomers and dimers

The SQUID and the ⁵⁷Fe Mössbauer spectroscopy studies of the magnetic properties of monomeric and dimeric forms of iron porphyrin were performed between 2 and 305 K. The effective magnetic relaxation rate of the Fe atoms in iron porphyrin monomers exhibits complex temperature dependence, resulting from the competing spin-spin and spin-lattice relaxation processes. The dimerization of iron porphyrin dramatically speeds up the magnetic relaxation. The Fe-Fe antiferromagnetic exchange coupling constant in Fe-O-Fe dimer is J≈−110 cm⁻¹. The complementary application of SQUID and the Mössbauer spectroscopy is proposed as a new precise quantitative analytical methodology for monitoring of the aggregation process of iron porphyrin.     

Studies on interpolymer self-organisation behaviour of protoporphyrin IX bound poly(carboxylic acid)s with complimentary polymers by means of fluorescence techniques

Covalently bound protoporphyrin IX was used as a fluorophore to investigate the interpolymer complex formation between the poly(carboxylic acid)s, PMAA/PAA and poly(N-vinyl pyrrolidone), PVP, poly(ethylene oxide), PEO or poly(ethylene glycol), PEG. Absorption and emission spectral properties of protoporphyrin IX bound to PAA, PMAA and PVP have been studied. Protoporphyrin IX in poly(MAA-co-PPIX) was found to be present in the dimer or higher aggregated form at low pH due to the environmental restriction imposed by the polymer whereas in the case of poly(AA-co-PPIX) and poly(VP-co-PPIX), PPIX exists in monomeric form. The fluorescence intensity and lifetime of PPIX bound to poly(carboxylic acid)s increase on complexation through hydrogen bonding with PVP, PEO and PEG due to the displacement of water molecules in the vicinity of the PPIX. Poly(MAA-co-PPIX) shows longer fluorescence lifetime due to the more compact interpolymer complexation as compared to poly(AA-co-PPIX) due to the enhanced hydrophobicity of PMAA. Poly(VP-co-PPIX) shows a decrease in the fluorescence lifetime on complexation with PMAA or PAA due to the hydrophilic and microgel like environment of the fluorophore bound to PVP. The contrasting behaviour of the same polymer adduct with respect to the site of the fluorophore is interpreted to be due to the solvent structure which determines the environment of the fluorophore.     

放射增敏性导弹型卟啉的合成与性质研究

用化学方法成功地将放射增敏剂灭滴灵连接到对癌细胞具有定位作用的原卟啉上，通过红外光谱、紫外可见吸收光谱、质谱、核磁共振谱和元素分析等手段确证了其结构。经动物实验证明，放射增敏效果较好。     

DHL细胞中5-ALA代谢PpIX的双光子荧光光谱

双光子激发生物组织荧光,激发光仅作用于焦点区域,对生物样品的光漂白性和光毒性都很小,因而双光子荧光显微技术已成为细胞生物学研究的一种新技术。文章采用波长为820 nm飞秒激光激发孵育有5-ALA的DHL细胞,在激光扫描显微镜的Lambda模式中获得单个DHL细胞的双光子荧光光谱,并测量DHL细胞内积聚的卟啉九(PpIX)特征荧光值。获得了浓度分别为2, 4和10 mmol·L-1的5-ALA溶液中,细胞代谢的PpIX含量随孵育时间的变化情况。DHL细胞内积聚的PpIX处于动态变化过程,并呈现出两阶段性的特点：细胞内积聚的PpIX含量随着孵育时间增长而增加,在3 h附近达到最大值,随后随着孵育时间增长反而下降。结果表明,基于激光扫描显微的双光子荧光光谱可成为DHL细胞等白血病细胞摄取5-ALA并生成PpIX的动力学研究的有效方法。     

Fabrication of a polypseudorotaxane nanoparticle with synergistic photodynamic and chemotherapy

Polypseudorotaxane (PPR) nanoparticles were fabricated by the self-assembly of mPEG-protoporphyrin IX (PpIX) conjugate and a-CDs via the hostguest interaction for achieving synergistic photodynamic and chemotherapy.     

Sonodynamic effects of protoporphyrin IX disodium salt on isolated sarcoma 180 cells

The cytotoxic effect of PPIX on isolated sarcoma 180 cells induced by ultrasound was investigated. Tumor cells suspended in air-saturated PBS (pH 7.2) were exposed to ultrasound at 2.2 MHz for up to 60 s in the presence and absence of protoporphyrin IX disodium salt (PPIX). The viability of cells was determined by a trypan blue exclusion test. The rate of ultrasonically induced cell damage was increased with 40–160 μM PPIX, while no cell damage was observed with 160 μM PPIX alone. This enhancement of cell damage with PPIX was inhibited by histidine. The participation of lipid peroxidation products in the cell damage process was also investigated. Scanning electron microscope (SEM) observation of the surface of cells was performed to evaluate the morphological changes induced by ultrasonic irradiation. The results indicate the involvement of a sonochemical mechanism.