奈米金粒子在光聲生醫影像之應用

Abstract

光聲影像為一新興之影像技術，在生醫應用上以得知生物組織中光學的特性為主。然而目前光聲影像研究大多仍侷限於型態觀察為主，而由於入射光能量在生物組織中強散射以及衰減皆會降低光聲影像之靈敏度，在此篇論文中，將使用金奈米粒子來增強光聲影像之偵測靈敏度以及擴展其應用範疇至功能性影像以及分子影像，其應用包含心血管疾病以及腫瘤之偵測以及診斷。在本論文的第一部份中，使用金奈米粒子作為指示劑，以實驗證明時間強度法量化估測血流速度之可行性。沖入法利用金奈米桿在適當脈衝雷射照射下產生形變的特性，實驗結果顯示沖出以及沖入兩種方法所測得之流速與實際流速之間之相關係數皆接近一，顯示其相對流速測量能力。在分子影像的應用上，使用與生化分子接合而具有特異性靶向能力之金奈米，可以偵測癌症細胞表面致癌基因的表現，以口腔癌細胞OECM1以及Cal27為測試細胞株。離體細胞實驗以及活體小動物實驗結果皆顯示，相較於與純金奈米桿反應，兩種癌細胞與各自有高表現之抗體接合金奈米桿反應能得到較強之光聲訊號，另外，與縮氨酸接合之金奈米桿也被用來對於腫瘤血管新生部份作特異性的辨識。而同時多重靶向性以得到多重分子表徵的目的，也以注射混合之奈米探針並以不同之波長雷射得到光聲影像驗證，注射混合探針之腫瘤在不同波長所得之影像強度皆大於注射純金奈米桿之腫瘤影像強度。靶向性熱治療也可藉由將一連續波雷射與光聲分子影像系統而達成，同時為了安全以及熱治療效力的考量，可在熱治療同時以光聲訊號即時且非侵入式的監控溫度變化，以所得光聲影像量化計算出的溫度影像顯示以加熱靶向性之金奈米桿方式可使腫瘤區域達到過高熱狀態，顯示可達到熱治療目的以及量化估測溫度之目的。腫瘤之病理切片結果也驗證靶向熱治療的效果，在未傷及正常細胞的情況下，腫瘤細胞可有效的被殺死。在本論文的最後，發展了一分頻影像方法，可有效提昇光聲影像之對比，此方法之根據為較高光吸收物質產生之光聲訊號含有較多的高頻成份，因此適當地選擇訊號分頻影像，可有效提昇影像之對比。以光吸收度相差八倍之吸收體所得影像之對比可藉由選擇較高頻之頻帶成像而有顯著的提昇。這些方法有助於提昇在高散射的活體實驗中對於含有金奈米粒子區域(因此光吸收度較高)之偵測能力。本研究使用金奈米實現了光聲功能性影像以及分子影像，包含定量血流量測以及腫瘤的多重靶向性。未來工作以活體之腫瘤血管新生區域流速量測以及提昇系統之靈敏度為主。

Photoacoustic imaging is a new imaging technique that has been used to image the optical properties of biological tissues. However, the applications of photoacoustic imaging in most researches have been limited to morphological observations. Also, high optical scattering of biological tissues and depth dependent decay of incident optical energy degrade the detection sensitivity. In this thesis, the use of gold nanoparticles as photoacoustic contrast agents helps to increase the sensitivity of photoacoustic imaging and to extend the applications to functional and molecular imaging, including cardiac functional assessment and cancer diagnosis. In the first part of the thesis, quantitative blood flow measurements based on the time-intensity method were tested. Gold nanoparticles were used as a photoacoustic contrast agent. In wash-in methods, the photo-induced rod-to-sphere shape transformation of gold nanorods was utilized. Results show that the correlation coefficients between the measured velocities and the true values are close to unity. For molecular imaging, photoacoustic imaging was utilized to probe information from oncogene surface molecules of oral cancer cell, OECM1 and Cal27, with the aid of bioconjugated gold nanorods. In vitro cell culture and in vivo small animal results show that both cancer cells with antibody conjugated nanorods of specific targeting exhibit a higher photoacoustic response than control groups with plain nanorods. Also, angiogenesis targeting was achieved with peptide conjugated gold nanorods. Images of OECM1 tumor with mixed nanoprobes injection also reveal enhanced photoacoustic intensity at different wavelengths than those with mixed plain gold nanorods injection, thus demonstrating simultaneous multiple targeting is feasible and can be used to obtain variable molecular signatures by simply switching laser wavelength. Also, photoacoustic pressure amplitude, which is linearly correlated with temperature, can be applied for real-time monitoring of the temperature non-invasively during photothermal therapy. Quantitative thermal imaging showed that the temperature can reach the hyperthermia level for effective cancer treatment. The results of pathological analysis of the tumor confirm the necrosis of the tumor cells without damaging surrounding normal cells. In the last part of the thesis, a subband imaging method to further enhance the image contrast was developed. The method was based on that high-absorption media generate acoustic waves with higher frequency components, and hence the imaging contrast can be enhanced by appropriate selection of the spectral subbands. The experimental results revealed that the contrast between two absorbers with eight times difference in optical absorption can be effectively increased by choosing a higher subband. These methods help to recognize the region of interest with gold nanoparticles particularly for in vivo studies. In summary, the thesis has realized photoacoustic functional and molecular imaging including quantitative flow/perfusion estimation and multiple targeting on tumor with the aid of gold nanoparticles. Future works will focus on in vivo flow estimation on tumor angiogenesis and improvement of detection sensitivity.