An apparatus was designed to provide a direct contact between a low-frequency ultrasound transducer and a hydrogel that contained a tracer nanoparticle ( Fig. 1). The low-frequency ultrasound was stimulated by a signal generator (40 kHz at 0.2 W/cm², marked as 5 in Fig. 1). The hydrogel was formed by dissolving agarose (0.5% w/v) in MilliQ water at a temperature above the gelling point, pouring the agarose solution into a mould and then allowing the solution to cool below the gelling temperature. The tracer gold nanoparticles (15-20 nm) were added to the agarose solution before it was allowed to cool below the gelling temperature. The gel formed with the incorporated gold nanoparticles was called the “tracer-gel” and is marked as 2 in Fig. 1. Another agarose gel was formed without adding any nanoparticles and this was called the “receiver-gel” and is marked as 3 in Fig. 1. The “tracer-gel” was firmly contacted to the ultrasound transducer (marked as 1 in Fig. 1). The combination of the ultrasound transducer, “tracer-gel” and “receiver-gel” were supported by a microscope slide ( Fig. 1).

The diffusion of the tracer gold nanoparticles was quantified by measuring the change in transmittance of the “receiver-gel” that was firmly contacted to the “tracer-gel”. The change in transmittance was measured by recording the output voltage of a photodiode (marked as 7 in Fig. 1) upon which the beam from a HeNe laser was directed (marked as 6 in Fig. 1). For example, the output voltage from the photodiode would decrease if the transmittance of the “receiver-gel” was reduced due to the accumulation of the tracer gold nanoparticles. The transmittance of the “receiver-gel” was measured at a known distance from the interface at the “tracer-gel” (marked as x in Fig. 1). In the experiments, the transmittance was measured at the distances of x = 1mm and x = 2mm. The transmittance data were captured by a computer (marked 8 in Fig. 1).

The principle of the in vitro system was tested by placing the “tracer-gel” in direct contact with the conjunctiva and sclera at the limbal region of the eye of a rabbit. In effect, the tissues at the limbal region substituted for the “receiver-gel”. The “tracer-gel” included therapeutic molecules that would usually have been injected directly into the vitreous humor of the eye in order for diffusion to the retina. The thickness of the tissues at the limbal region of the eye is around 0.5mm or less, so the hypothesis was that the ultrasound would drive the therapeutic molecules through the conjunctiva and sclera to be delivered to the capillary circulation of the uveal tract in the region of the pars plana. The uveal circulation would then deliver the therapeutic molecules to the interior of the eye, including to the retina.

Therapeutic molecules were loaded into the hydrogel (“tracer-gel”) that was attached to the ultrasound transducer. The therapeutic drugs replaced the gold nanoparticles that were used for the in vitro experiments. The therapeutic molecules were bevacizumab (25 mg/ml), ranibizumab (3 mg/ml) and verteporfin (2 mg/ml) either incorporated directly into the agarose “tracer-gel” or encapsulated in liposomes that were then incorporated into the agarose “tracer-gel”. Each drug was incorporated in the “tracer-gel” at the concentration used for the current clinical procedure of direct injection into the vitreous chamber (bevacizumab, ranibizumab) or IV injection (verteporfin).

(a) Encapsulation of drug into liposomes

The principle was to prepare a dried film of lipid, rehydrate the lipid in a buffer containing the drug to be packaged and then produce homogeneous liposomes of 1μm diameter using an extrusion procedure. The result of that procedure was a 1ml volume of a concentrated solution of liposomes of 1μm diameter that were loaded with the drug. This was achieved using the following procedure. A quantity of 40μg of L-α-lecithin (Sigma, P-5638) was added to a glass tube and 400ml of chloroform was added to the glass tube in order to achieve a lipid concentration of 100mg/ml and vortex mixed to ensure the lipid was completely dissolved. The lipid/chloroform mixture was dried under a stream of N₂ gas to remove the volatile chloroform. The glass tube was left under vacuum for at least 12 hours at room temperature in order to remove all traces of chloroform. Then a 1ml solution of the therapeutic drug, at the required concentration, was addd to the glass tube and vortex mixed for 5 minutes to ensure complete solution of the lipid. The tube was left to stand at room temperature for at least 15 minutes, then vortex mixed again for 5 minutes. This mixture was transferred to a syringe for extrusion through a 1μm polycarbonate filter 19 times in order to produce a homogeneous solution of liposomes. The liposome mixture was transferred to an Eppendorf tube and centrifuged at 9,000rpm for 30 minutes in order to pellet the liposomes, the supernatant was removed and the pellet resuspended in a clear 150mM NaCl (unbuffered) solution. This final solution ws incorporated into the agarose “tracer-gel”.

Figure 1.

Apparatus used to measure the transport of tracer gold nanoparticles that as stimulated by low-frequency ultrasound. The block diagram of the components is drawn in panel A, and the actual gels+transducer is shown in panel B. The light path is illustrated with the dotted line, and was aimed to pass through the “receiver-gel” at a distance marked x below the interface with the “tracer-gel”. The numbers in panels A and B refer to the same components. (1=ultrasonic transducer, 2=”tracer-gel”, 3=”receiver-gel”, 4=glass microscope slide, 5=signal generator, 6=HeNe laser, 7=photodiode, 8=computer).

This experiment was conducted in 2 parts with gold tracer nanoparticles incorporated in the “tracer-gel”, which were (i) measurement of the transmittance in the “receiver-gel” at a distance of x=1mm below the interface, and (ii) measure of the transmittance in the “receiver-gel” at a distance of x=2mm below the interface. A negative control experiment was conducted to confirm whether changes in transmittance were due to transport of potential degradation products of the polymers as an explanation for any change in transmittance induced by the ultrasound. In that control experiment, the “tracer-gel” did not contain any gold nanoparticles and the transmittance in the “receiver-gel” was measured at a distance of x=1mm below the interface. The interface is defined as the boundary between block 2 and block 3 in Fig. 1. In all these experiments the photodiode output (mV) in the graphs indicates the transmittance. An increase in the transmittance of the gel corresponds to an increase in the photodiode output and a decrease in the transmittance of the gel corresponds to a decrease in the photodiode output.

The measured transmittance in the “receiver-gel” at x=1mm below the interface is shown in Fig. 2. The diffusion of gold nanoparticles into the “receiver-gel” is measured by the change in transmittance. For example, we expect that an increase in the amount of gold nanoparticles diffusing into the clear gel would result in a decrease in the transmittance of the clear gel.

Figure 2.

Measurement of transmittance of the “receiver-gel” at x=1mm depth from the interface with the “tracer-gel” that contained the gold nanoparticles.

Figure 3.

Measurement of transmittance of the “receiver-gel” at x=2mm depth from the interface with the “tracer-gel” that contained the gold nanoparticles.

Fig. 2 illustrates that the 40kHz ultrasound stimulated the diffusion of the gold nanoparticles from the “tracer-gel” into the “receiver-gel”. There are 3 distinct regions of the data in Fig. 2: During the period from 0-10 minutes without the ultrasound switched on the transmittance of the “receiver-gel” was at a plateau level. The fluctuations may have been due to the initial setting up of the experiment. However, the most important feature is that the average level of transmittance was not changing. During the period from 10-30 minutes the 40kHz ultrasound was switched on, as indicated by the arrows. The transmittance of the gel decreased (photodiode output decreased) soon after the 40kHz ultrasound was switched on. The decrease in transmittance was rapid at first, and then reached a plateau after about 18 minutes. The plateau phase continued until the 40kHz ultrasound was switched off. The plateau was probably due to the gold nanoparticles only being able to diffuse, under the stimulation of the ultrasound, for a limited distance into the clear gel. In this case, the distance was 1 mm. This limit on diffusion can be explained by the mass of the gold nanoparticles, and comparison with measurements from techniques such as gel electrophoresis. For example, in gel electrophoresis the diffusion of substances is enhanced by an electrical current applied to a gel. A substance will diffuse more if it has a smaller mass, with substances of heavier mass diffusing less distance into the gel. By analogy, in the case of the 40kHz ultrasound the gold nanoparticles diffused as far into the “receiver-gel” as their mass would allow. During the period from 30-45 minutes the transmittance remained at the plateau level after the 40 kHz ultrasound was switched off.

This limitation on how far the gold nanoparticle could diffuse into the gel is illustrated by the data shown in Fig. 3. The data in Fig. 3 are of the transmittance of the “receiver-gel” at a distance of x=2mm below the interface. The transmittance in Fig. 3 does not change when the 40kHz was turned on. The small “bump” at around 20minutes is most likely due to an artefact. This lack of change in transmittance follows from the above explanation, whereby the mass of the gold nanoparticles is too great to allow the ultrasound energy at 40kHz to provide enough force (momentum) to enhance the diffusion of the gold nanoparticles further than about 1mm into the “receiver-gel”.

To support those interpretations of the experiment, the “receiver-gel” was imaged by scanning electron microscopy (SEM) and the particles that were observed in the SEM images were analysed by energy dispersive spectroscopy (EDS). Fig. 4 shows that the “receiver-gel” contained electron-dense particles. These particles are the lighter grey discrete objects resting on the underlying dehydrated gel. The location of these particles was at the surface of the dehydrated gel, which implied that the particles were at or just beneath the surface of the hydrated gel as it was used in the experiment.

Several of the particles in Fig. 4 appear to be quite large, and approximately 5mm in size. This is much larger than the size of the gold nanoparticles used as the tracer. However, there are also smaller electron-dense particles in the clear gel, which are of the appropriate size (indicated by arrows in Fig 4). The chemical composition of all the particles was analysed by EDS. The larger particles contained C, O, Na, Mg, Si, Cl, Ca, Zn, Fe and Cu. All of those chemicals are present in the buffer solutions used for the experiment. Thus, the larger particles are most likely crystals of the chemicals used for the solutions used to hydrate the gels.

Fig. 5 illustrates that the chemical composition of the smaller particles was gold. It is common for the gold nanoparticles to form clusters, which accounts for the size of the smaller particles as shown in Fig. 5.

A negative control experiment was conducted to confirm whether ultrasound-induced changes in transmittance could be explained by the transport of potential degradation products of the polymers. In that control experiment, the “tracer-gel” did not contain any gold nanoparticles and the transmittance in the “receiver-gel” was measured at a distance of x=1mm below the interface. The results for this control experiment are shown in Fig. 6, which show that the transmittance of the “receiver-gel” did not change when measured at a distance of x=1mm from the interface. This “receiver-gel” was analysed using SEM/EDS and no gold particles were found (data not shown).

This (negative) control experiment provided supporting evidence that the diffusion of the gold nanoparticles was enhanced by the 40kHz ultrasound and that the gold nanoparticles could only diffuse 1mm into the “receiver-gel”.

Figure 4.

SEM image of the “receiver-gel” from the diffusion experiment shown in Fig. 1. For the purposes of the SEM imaging the gel was dehydrated. The electron-dense particles (light grey colour) are at the surface of the dehydrated gel. The arrows point to particles that are most likely to be gold, with the alrger particles most likely to be crystals of chemicals used in the buffer solutions (see text for explanation).

Figure 5.

SEM/EDS analysis of the smaller particles observed in the dehydrated gels shown in Figure 4. The EDS analysis in panel B is of the particle imaged by SEM in panel A. The analysis shows that the composition of the particle in panel A is gold.

Fig. 7 shows sections of an eye stained with Masson stain. The sections were from an eye into which bevacizumab was delivered from the “tracer-gel”. The sections are useful for orientation, with collagen stained blue (cornea, sclera) and neural retina, epithelium and muscle stained crimson. The embedding and sectioning procedures have caused the retina to be dislodged from its normal position in contact with the sclera.

Fig. 8 shows sections of the retina around the posterior pole of the eyes of the 3 rabbits. The green fluorescence is not intense because of the very stringent blocking (donkey serum, overnight at 4°C) and slow antibody reaction (overnight at 4°C) to minimise artefacts. The main point to note is the presence of the green staining in the outer retina and choriocapillaris in the images of eyes where bevacizumab ( Fig. 8A, 8B), ranibizumab ( Fig. 8D) and verteporfin (Fig. 8F) were delivered by the ultrasound stimulation. The outer retina is usually connected to the scleral tissue, but in these sections the retina had separated from the sclera. In comparison the negative control eye (“tracer-gel” with no drug) shows no green fluorescence ( Fig. 8C). The positive control eye (direct injection into the vitreous chamber) shows green fluorescence ( Fig. 8E).

Figure 6.

Measurement of transmittance of the “receiver-gel” at x=1mm depth from the interface with the “tracer-gel” that did not contain any gold nanoparticles.

Figure 7A.

Masson-stained section of a posterior area of a rabbit eye into which bevacizumab was delivered from the“tracer-gel”. The sclera is stained bright blue and the retina, epithelia and muscle stained crimson. The retina and choroid have separated from the sclera.

Figure 7B.

Masson-stained section of the limbal region of a rabbit eye into which bevacizumab was delivered from the “tracer-gel”. The sclera and cornea are stained bright blue and the retina, epithelia and muscle stained crimson. The retina and choroid have separated from the sclera.