Final Report Summary - SMARTPTDRUGS (Fluorescent nanocrystals for activation and delivery of platinum drugs)
In this project, we wished to explore if we could use nanocrystals made of semiconductor material to control the generation and release of Pt(II) anticancer drugs (e.g. cisplatin, one of the most powerful and widely used anticancer drugs in the clinic) using visible or near infra-red light. These materials are called quantum dots (QDs) and have exceptional fluorescent and electronic properties. These properties have already made these materials tremendously attractive in other important applications - as transistors, in solar cells, light-emitting diodes (LEDs), in biological / medical imaging etc. We wanted to exploit the unique physicochemical properties of QDs also for tumour imaging (detection) purposes.
The research project has required the development/execution of five different tasks as it was described in the proposal. Although most of these tasks have been developed and carried out simultaneously, the fellow (Dr Maldonado) has been fundamentally focused on the first three tasks during the first period (October 2010 - September 2011) and in the last two tasks during the second period (October 2011 - September 2012). The work and main achievements accomplished during this project are summarised below.
Task 1: Preparation and characterisation of size-selected QDs with suitable semiconductor material.
Several hydrophobic core-shell CdSe@ZnS QDs were synthesised and purified adapting. QDs containing CdS proved to be a reasonable choice for this proof-of-concept project as the reduction potential for these photoexcited QDs is more negative (ECB 1 V vs NHE) than the reduction potential of most of the Pt(IV) complexes (ranging from 0 to 1 V), which we wanted to use as anticancer prodrugs. Moreover, it has been previously reported that ZnS overcoating typically leads to core-shell QDs which are more stable, more strongly fluorescent and less toxic. Using the hot-injection technique, Dr Maldonado has been able to prepare core-shell CdSe@ZnS QDs of different sizes and therefore emission wavelengths (up to 640 nm).
Of particular interest is the use of longer wavelength light (near infrared region) to photoactivate the Pt(IV) prodrugs as it penetrates tissue more deeply, and therefore is ideal for biomedical applications. With this idea in mind and looking for longer-term biocompatible QDs, she has also synthesised and characterised core-shell Cd-free QDs CuInS2@ZnS emitting from 560 to 710 nm, which was not initially considered in this proposal. Based on recent developments these novel QDs should prove to be more desirable for long term applications of the technology (i.e. in a pre-clinical and even clinical setting) because of they have low toxicity.
All synthesised QDs were characterised by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), ultraviolet-vis and fluorescence spectroscopy, nuclear magnetic resonance (NMR), dynamic light scattering (DLS) and inductive coupled plasma - atomic emission spectroscopy (ICP-AES).
Task 2: Achieving / optimising water solubility, cell internalisation, tumour targeting and interaction with Pt(IV) complex through QD surface modification / bioconjugation. This second task was complex and maybe the most critical one. It involved identifying and optimising the surface ligands, tumour targeting, and nature of the interaction with the Pt(IV) complex for selectively killing cancer cells only after irradiation of light of a specific wavelength. For this reason, the fellow worked extensively on this task throughout the entire project.
The synthesised QDs in task 1 (CdSe@ZnS and CuInS2@ZnS) initially were hydrophobic and therefore insoluble in water and not suitable for biomedical applications. Different strategies were pursued to make these QDs soluble in water: (i) the use of bi-functional ligands such as dimercapto succinic acid (DMSA) to substitute the hydrophobic ligands on the surface of the nanoparticle and (ii) the creation of micelles with encapsulated QDs (MQDs) inside prepared by self-assembly of phospholipids with PEG 2000 around the hydrophobic QDs. Of these two strategies, the second proved to be more versatile, yielding closely packed QDs with a hydrodynamic size < 50 nm, which are stable in deionised water over a wide pH range of 1 - 10 and in PBS for over a month. These MQDs were then characterised by techniques such as TEM, XPS, ultraviolet-vis and fluorescence spectroscopy, NMR, ICP-AES and DLS.
Regarding to cell internalisation and tumour targeting, she found in the literature interesting peptide sequences (SWL and YSA) which are mimetic of ephrin-A1 (Biochemistry, 2010, 49, 6687) and therefore target the EphA2 receptor. This tyrosine kinase receptor (EphA2) is a protein membrane which has recently emerged as a promising new therapeutic target in cancer because of its high level of expression in tumours (including breast, ovarian, prostate, pancreatic, and lung cancer). In fact, one of these ephrin-A1 mimetic peptides has been recently conjugated to superparamagnetic nanoparticles and used successfully to capture and remove cultured human ovarian cancer cells from the peritonea of experimental mice (Nanomedicine: Nanotechnology, Biology, and Medicine, 2010, 6, 399).
Encouraged by these promising results, she attached several ephrin-A1 mimetic peptide sequences to the surface of different nanoparticles (size and composition). Although the attachment of these peptides was successful, no significant enhancement in the cellular uptake of these labeled nanoparticles in different cancer cell lines overexpressing the EphA2 receptor was found (studied by confocal microscopy).
Then, Dr Maldonado decided to conjugate the natural ligand (ephrin-A1) directly to the nanoparticle through covalent interaction between the carboxylic groups of PEG-COOH moieties and amine groups of the ephrin-A1 ligand. The conjugation, using EDC and NHSS as coupling agents, was successful (around 15 ephrin-A1 units per nanoparticle were attached). In this case, the ephrin-A1 ligands kept their activity (confirmed by Western Blot). Recently, we started to attach other targeting vectors for which there is more information in the literature such as folic acid and RGD peptides to do a comparative study. Also, in collaboration with Dr Valerie Brunton at the Edinburgh Cancer Research UK Centre we are exploring the importance of the surface ligands, shape and sizes of the QDs prepared by Dr Maldonado in reaching and penetrating the tumour.
Task 3: Synthesis of Pt(IV) complexes
During the two years, the fellow synthesised a wide battery of Pt(IV) complexes with different hydrophobicity, charge and redox potential. All the Pt(IV) complexes were characterised by elemental analysis, NMR, Mass Spectroscopy (MS) and FT-IR spectroscopy.
It was considered that the ideal Pt(IV) complex candidate would be one which had high solubility in water, was not toxic, had a relatively high reduction potential (that is, difficult to reduce by biological reducing agents and therefore more stable in the bloodstream) and with axial ligands with functional groups which could potentially be used to attach targeting biomolecules.
Amongst all these new Pt(IV) complexes, she found that the succinic cisplatin derivative cis, cis, trans-(Pt(NH3)2Cl2(O2CCH2CH2CO2H)2) all of these required conditions/criteria and therefore was ideal for further studies (including in vitro and photochemical studies). This complex was prepared by reaction of succinic anhydride with cis, cis, trans-(Pt(NH3)2Cl2(OH)2) prepared by the oxidation of cis-(Pt(NH3)2Cl2) with hydrogen peroxide. The complex was high soluble in water (up to mM concentration), non-toxic (IC50 around 500uM on a human prostate cancer cell line, PC3), its reduction potential was relatively high and the carboxylic groups of the succinic moieties were available to attach it to the nanoparticle or to link bioactive molecules such as folate, small peptide sequences, etc.
Task 4: Photochemistry and metal complex speciation
The photochemical reactions between the QDs and the selected Pt(IV) candidate were followed by NMR spectroscopy. The 1H NMR spectrum of the Pt(IV) prodrug showed two sets of multiplets in water due to two sets of CH2 protons. In the presence of MQDs, the peaks were slightly upfield shifted, presumably by the interaction with the MQD. When mixtures of MQD (40nM) and the Pt(IV) complex (50, 100, 200, 300, 500 uM and 1 mM) were irradiated with a low power LED source (480 nm, 14 mW / cm2), a singlet due to succinate appeared due to loss of the axial ligands, which increases with an increase in the concentration of the Pt(IV) complex. In the absence of MQDs the amount of succinate was very small even after 5 h irradiation (< 5 %). To elucidate the oxidation state of the platinum complex generated after irradiation with visible light, the fellow carried out XPS studies at the Pt 4f region. The ratio Pt(II) / Pt(IV) calculated by XPS was identical to the ratio calculated by proton (1H) NMR using the succinate peak, which is indicative of reduction of Pt(IV) to Pt(II) by the photoinduced electron transfer mechanism. As expected from this mechanism, the fluorescence of the MQDs was quenched in the presence of the Pt(IV) complex. Dr. Maldonado has found similar results for micelles encapsulating CdSe@ZnS and CuInS2@ZnS and when the mixtures were irradiated with other visible wavelengths. In general, the results showed that the platinum concentration plays the critical role in the photoreduction process. Therefore, it seems clear that by conjugated/incorporated the Pt(IV) prodrug in a good yield onto the surface of the nanoparticle the results can be improved even further (future work). At the moment, the host group is working on developing this aspect of the project further.
TASK 5: Cell imaging and cytotoxicity studies
To evaluate the feasibility of using MQDs for delivering a toxic dose of platinum drugs only in the presence of light, the fellow conducted in vitro cytotoxicity assays of this Pt(IV) prodrug candidate, MQD-Pt(IV) complex mixtures and MQDs prior and following irradiation with light on the human prostate cancer cell line PC3. Although some reports have found some QDs toxic, no significant toxicity of the MQDs was observed in this study in the low concentrations required to photogenerate cisplatin. This result is consistent with previous in vitro and in vivo studies in which the QD toxicity is reduced in micellar and liposomal QD constructs. When the Pt(IV) complex was irradiated with light prior to incubation with the cells its cytotoxicity did not increase significantly due to poor photoinduced conversion to cytotoxic Pt(II) species. However, when this was done in the presence of MQDs in nanomolar concentrations the cytotoxicity was significantly enhanced (IC50 25 M).
This is the first example of QD and light induced generation of cisplatin from a Pt(IV) complex, and opens ups new opportunities in nanomedicine and platinum-based therapy.
As part of the project the fellow also contributed to the development of new sensors against toxic substances, multimodal contrast agents (agents to visualise biological subjects in vitro and in vivo using complementary techniques) and DNA cleaving agents based on QDs. All of these results have impact and applications in different fields (environmental safety monitoring, biotechnology, cancer diagnosis, cancer treatment etc.).
The combination of imaging with externally activated therapy (theranostic systems) which the fellow achieved with MQDs and Pt(IV) prodrugs could lead to (after further studies and development) diagnosing, imaging and treating cancer with high spatial and temporal resolution and control, and therefore for detecting disease earlier and applying chemotherapy with fewer side effects and saving money. Thus, overall this has been a project which will inspire new ways of detecting and treating cancer with higher efficiency and fewer side effects and costs (the host group has secured funding to extend/continue the work).
The research project has required the development/execution of five different tasks as it was described in the proposal. Although most of these tasks have been developed and carried out simultaneously, the fellow (Dr Maldonado) has been fundamentally focused on the first three tasks during the first period (October 2010 - September 2011) and in the last two tasks during the second period (October 2011 - September 2012). The work and main achievements accomplished during this project are summarised below.
Task 1: Preparation and characterisation of size-selected QDs with suitable semiconductor material.
Several hydrophobic core-shell CdSe@ZnS QDs were synthesised and purified adapting. QDs containing CdS proved to be a reasonable choice for this proof-of-concept project as the reduction potential for these photoexcited QDs is more negative (ECB 1 V vs NHE) than the reduction potential of most of the Pt(IV) complexes (ranging from 0 to 1 V), which we wanted to use as anticancer prodrugs. Moreover, it has been previously reported that ZnS overcoating typically leads to core-shell QDs which are more stable, more strongly fluorescent and less toxic. Using the hot-injection technique, Dr Maldonado has been able to prepare core-shell CdSe@ZnS QDs of different sizes and therefore emission wavelengths (up to 640 nm).
Of particular interest is the use of longer wavelength light (near infrared region) to photoactivate the Pt(IV) prodrugs as it penetrates tissue more deeply, and therefore is ideal for biomedical applications. With this idea in mind and looking for longer-term biocompatible QDs, she has also synthesised and characterised core-shell Cd-free QDs CuInS2@ZnS emitting from 560 to 710 nm, which was not initially considered in this proposal. Based on recent developments these novel QDs should prove to be more desirable for long term applications of the technology (i.e. in a pre-clinical and even clinical setting) because of they have low toxicity.
All synthesised QDs were characterised by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), ultraviolet-vis and fluorescence spectroscopy, nuclear magnetic resonance (NMR), dynamic light scattering (DLS) and inductive coupled plasma - atomic emission spectroscopy (ICP-AES).
Task 2: Achieving / optimising water solubility, cell internalisation, tumour targeting and interaction with Pt(IV) complex through QD surface modification / bioconjugation. This second task was complex and maybe the most critical one. It involved identifying and optimising the surface ligands, tumour targeting, and nature of the interaction with the Pt(IV) complex for selectively killing cancer cells only after irradiation of light of a specific wavelength. For this reason, the fellow worked extensively on this task throughout the entire project.
The synthesised QDs in task 1 (CdSe@ZnS and CuInS2@ZnS) initially were hydrophobic and therefore insoluble in water and not suitable for biomedical applications. Different strategies were pursued to make these QDs soluble in water: (i) the use of bi-functional ligands such as dimercapto succinic acid (DMSA) to substitute the hydrophobic ligands on the surface of the nanoparticle and (ii) the creation of micelles with encapsulated QDs (MQDs) inside prepared by self-assembly of phospholipids with PEG 2000 around the hydrophobic QDs. Of these two strategies, the second proved to be more versatile, yielding closely packed QDs with a hydrodynamic size < 50 nm, which are stable in deionised water over a wide pH range of 1 - 10 and in PBS for over a month. These MQDs were then characterised by techniques such as TEM, XPS, ultraviolet-vis and fluorescence spectroscopy, NMR, ICP-AES and DLS.
Regarding to cell internalisation and tumour targeting, she found in the literature interesting peptide sequences (SWL and YSA) which are mimetic of ephrin-A1 (Biochemistry, 2010, 49, 6687) and therefore target the EphA2 receptor. This tyrosine kinase receptor (EphA2) is a protein membrane which has recently emerged as a promising new therapeutic target in cancer because of its high level of expression in tumours (including breast, ovarian, prostate, pancreatic, and lung cancer). In fact, one of these ephrin-A1 mimetic peptides has been recently conjugated to superparamagnetic nanoparticles and used successfully to capture and remove cultured human ovarian cancer cells from the peritonea of experimental mice (Nanomedicine: Nanotechnology, Biology, and Medicine, 2010, 6, 399).
Encouraged by these promising results, she attached several ephrin-A1 mimetic peptide sequences to the surface of different nanoparticles (size and composition). Although the attachment of these peptides was successful, no significant enhancement in the cellular uptake of these labeled nanoparticles in different cancer cell lines overexpressing the EphA2 receptor was found (studied by confocal microscopy).
Then, Dr Maldonado decided to conjugate the natural ligand (ephrin-A1) directly to the nanoparticle through covalent interaction between the carboxylic groups of PEG-COOH moieties and amine groups of the ephrin-A1 ligand. The conjugation, using EDC and NHSS as coupling agents, was successful (around 15 ephrin-A1 units per nanoparticle were attached). In this case, the ephrin-A1 ligands kept their activity (confirmed by Western Blot). Recently, we started to attach other targeting vectors for which there is more information in the literature such as folic acid and RGD peptides to do a comparative study. Also, in collaboration with Dr Valerie Brunton at the Edinburgh Cancer Research UK Centre we are exploring the importance of the surface ligands, shape and sizes of the QDs prepared by Dr Maldonado in reaching and penetrating the tumour.
Task 3: Synthesis of Pt(IV) complexes
During the two years, the fellow synthesised a wide battery of Pt(IV) complexes with different hydrophobicity, charge and redox potential. All the Pt(IV) complexes were characterised by elemental analysis, NMR, Mass Spectroscopy (MS) and FT-IR spectroscopy.
It was considered that the ideal Pt(IV) complex candidate would be one which had high solubility in water, was not toxic, had a relatively high reduction potential (that is, difficult to reduce by biological reducing agents and therefore more stable in the bloodstream) and with axial ligands with functional groups which could potentially be used to attach targeting biomolecules.
Amongst all these new Pt(IV) complexes, she found that the succinic cisplatin derivative cis, cis, trans-(Pt(NH3)2Cl2(O2CCH2CH2CO2H)2) all of these required conditions/criteria and therefore was ideal for further studies (including in vitro and photochemical studies). This complex was prepared by reaction of succinic anhydride with cis, cis, trans-(Pt(NH3)2Cl2(OH)2) prepared by the oxidation of cis-(Pt(NH3)2Cl2) with hydrogen peroxide. The complex was high soluble in water (up to mM concentration), non-toxic (IC50 around 500uM on a human prostate cancer cell line, PC3), its reduction potential was relatively high and the carboxylic groups of the succinic moieties were available to attach it to the nanoparticle or to link bioactive molecules such as folate, small peptide sequences, etc.
Task 4: Photochemistry and metal complex speciation
The photochemical reactions between the QDs and the selected Pt(IV) candidate were followed by NMR spectroscopy. The 1H NMR spectrum of the Pt(IV) prodrug showed two sets of multiplets in water due to two sets of CH2 protons. In the presence of MQDs, the peaks were slightly upfield shifted, presumably by the interaction with the MQD. When mixtures of MQD (40nM) and the Pt(IV) complex (50, 100, 200, 300, 500 uM and 1 mM) were irradiated with a low power LED source (480 nm, 14 mW / cm2), a singlet due to succinate appeared due to loss of the axial ligands, which increases with an increase in the concentration of the Pt(IV) complex. In the absence of MQDs the amount of succinate was very small even after 5 h irradiation (< 5 %). To elucidate the oxidation state of the platinum complex generated after irradiation with visible light, the fellow carried out XPS studies at the Pt 4f region. The ratio Pt(II) / Pt(IV) calculated by XPS was identical to the ratio calculated by proton (1H) NMR using the succinate peak, which is indicative of reduction of Pt(IV) to Pt(II) by the photoinduced electron transfer mechanism. As expected from this mechanism, the fluorescence of the MQDs was quenched in the presence of the Pt(IV) complex. Dr. Maldonado has found similar results for micelles encapsulating CdSe@ZnS and CuInS2@ZnS and when the mixtures were irradiated with other visible wavelengths. In general, the results showed that the platinum concentration plays the critical role in the photoreduction process. Therefore, it seems clear that by conjugated/incorporated the Pt(IV) prodrug in a good yield onto the surface of the nanoparticle the results can be improved even further (future work). At the moment, the host group is working on developing this aspect of the project further.
TASK 5: Cell imaging and cytotoxicity studies
To evaluate the feasibility of using MQDs for delivering a toxic dose of platinum drugs only in the presence of light, the fellow conducted in vitro cytotoxicity assays of this Pt(IV) prodrug candidate, MQD-Pt(IV) complex mixtures and MQDs prior and following irradiation with light on the human prostate cancer cell line PC3. Although some reports have found some QDs toxic, no significant toxicity of the MQDs was observed in this study in the low concentrations required to photogenerate cisplatin. This result is consistent with previous in vitro and in vivo studies in which the QD toxicity is reduced in micellar and liposomal QD constructs. When the Pt(IV) complex was irradiated with light prior to incubation with the cells its cytotoxicity did not increase significantly due to poor photoinduced conversion to cytotoxic Pt(II) species. However, when this was done in the presence of MQDs in nanomolar concentrations the cytotoxicity was significantly enhanced (IC50 25 M).
This is the first example of QD and light induced generation of cisplatin from a Pt(IV) complex, and opens ups new opportunities in nanomedicine and platinum-based therapy.
As part of the project the fellow also contributed to the development of new sensors against toxic substances, multimodal contrast agents (agents to visualise biological subjects in vitro and in vivo using complementary techniques) and DNA cleaving agents based on QDs. All of these results have impact and applications in different fields (environmental safety monitoring, biotechnology, cancer diagnosis, cancer treatment etc.).
The combination of imaging with externally activated therapy (theranostic systems) which the fellow achieved with MQDs and Pt(IV) prodrugs could lead to (after further studies and development) diagnosing, imaging and treating cancer with high spatial and temporal resolution and control, and therefore for detecting disease earlier and applying chemotherapy with fewer side effects and saving money. Thus, overall this has been a project which will inspire new ways of detecting and treating cancer with higher efficiency and fewer side effects and costs (the host group has secured funding to extend/continue the work).