Borosilicate glass capillaries femtotip II coated with silver is shown in the SEM image in Fig. , while Fig. shows the tip with the grown ZnO NRs. As can be seen, the ZnO NRs have a hexagonal shape and a uniform density as shown in SEM image of Fig. .
Images taken by a coupled charge camera device (CCD) connected to the inverted microscope are shown in Fig. . As a control experiment, the excitation of a bare and conjugated ZnO NRs was first performed. Figure , b shows the fluorescence from the tip of the PDT devices for cases with and without the protoporphyrin, respectively. These photographs were taken when a filter (DAP1) for UV excitation was used. It can be seen that the fluorescence is relatively high using both the bare ZnO and conjugated ZnO nanorods. As mentioned above, vertical nanorods are like natural wave guiding cavities for making the emitted light travels efficiently to the top of the device minimizing partial leakage and thus enhancing the light extraction efficiency from the PDT device [8
]. ZnO nanorods with a large surface area to volume ratio are of potential for more drug delivery to the tumor site. Different stages from the start of the mechanical manipulation of the PDT device inside the cancer cell until the cell necrosis are shown in Fig. –. Images shown in Fig. – show the tip and the cancer cell in the Petri dish. In the image shown in Fig. , the tip was near the cell but has not yet been inserted inside the cell. Here, the UV light exposure time was 10 min and there was no cell necrosis. It means neither the PPDME nor the molecular oxygen was available inside the cell to facilitate the production of singlet oxygen [20
]. Then we inserted the tip of the PDT device inside the cell as shown in the images displayed in Fig. , and the magnified images shown in Fig. , . It is observed that the tumor cell is a rapidly dividing cell, with destroyed outer barriers as shown in the image of Fig. . These are the basic properties of tumor cell along with other such as altered enzymes and increased blood perfusion [26
]. Then the cancer cell is exposed to UV environment for 10 min while the PDT device is inserted inside the cell. This is shown in the case displayed by the image in Fig. . In Fig. , the tip has been prepared as mentioned above with ZnO NRs conjugated with protoporphyrin. The basic needs for PDT is the availability of the molecular oxygen, through a photosensitizer excited by white light, which in our case can be emitted by ZnO nanorods [20
]. Then there is an energy exchange process between the conjugated tip and the molecular oxygen inside the cell. The phototoxic reaction involves the formation of singlet oxygen in the cell which causes initial damage to mitochondria leading to cell necrosis as shown in image of Fig. [22
]. The image shown in Fig. displays only tip of the PDT device after cell necrosis. The tip appears thicker after the manipulation inside the cell because some remnants’ of the cell are attached to it. We used a light exposure time of 10 min in many similar different repetition experiments and found similar result. It is noted that the tip of the present PDT device can be used only once for these local single cell experiment. The reason is that all the remnants of the cell i.e. calcium, protein, etc., which are parts of the membrane, stick on the tip and they will decrease the intensity of light used for cell necrosis. The control experiment using a bare ZnO NRs tip (without the PPDME) was also performed. Similar UV excitation light exposure time of 10 min was used. No immediate cell necrosis was observed as expected due to the absence of the PPDME.
Figure 2 Digital images taken during the performance of the intracellular measurements, a and b show the fluorescence images of the bare and the PPDME-conjugated ZnO NRs, respectively. c Image of the PPDME-conjugated ZnO NRs tip inside the cancer cell. d Image (more ...)
Figure shows a schematic diagram of the PDT mechanism involved in the present study. The mechanism starts with the excitation of the photosensitizer inside the cancer cell and the release of active species of oxygen which then leads to cellular toxicity and finally cell necrosis. ZnO NRs are excited by the UV and consequently emits white light which is absorbed by the PPDME (630 nm) [19
]. In turn, singlet oxygen is released to kill the mitochondria causing finally the required cell necrosis.
Schematic diagram showing the intracellular PDT process of cancer cell
Fluorescence spectrum (Fl) of both the bare and the conjugated PDT devices was also measured to identify the emission nature. Figure shows the Fl of the bare ZnO NRs-based PDT device measured at room temperature. This Fl spectrum shows five different peaks. These are the UV and the rest belong to the white light constituents. The first dominant Fl peak was observed at 337 nm and the origin of this peak is to further be investigated. The second peak at 395 nm is attributed to the recombination of free excitons in ZnO i.e. band edge emission [27
]. The third blue emission peak at 468 nm comes from the combination of zinc interstitial level to valence band combined with emission due to transmission from the zinc interstitial to zinc vacancies in ZnO [28
]. The fourth green emission 560 nm is attributed to a recombination of electrons at the conduction band with holes trapped in oxygen- and zinc-related defects [29
]. The fifth peak is at 680 nm is attributed to other deep level defect-related radiative transition in ZnO [28
]. The stronger intensity of the blue emission relative to the UV emission can suggest the non-stochiometric composition of ZnO [30
]. Figure shows the fluorescence emission of PDT-conjugated ZnO NRs device. Basically, it is consisting of the same peaks, except a small change of the nature probably due to the absorption by the PPDME. Moreover, the intensity of the Fl in Fig. is five times less than that of the Fl spectrum in Fig. indicating absorption of some of the emitted white light by the PPDME as required. From the combined results shown in Figs. and , we concluded that white light emission from ZnO was transferred by absorption to the PPDME, which in turn caused excitation and production of singlet oxygen which consequently caused the aimed cell necrosis.
Florescence (Fl) spectra of ZnO NRs, without (a) and (b) with protoporphyrin, respectively. These spectra were taken using a laser line of wavelength of 240 nm as an excitation source from a PTI-Fluorescence system