Final Report Summary - PINSYS (Bio-Inspired Approaches to Porous Inorganic Nanoparticles and Their Application as Targeted Drug Delivery Systems)
Overview: Biominerals, with their hierarchical, composite structures and complex morphologies provide a unique inspiration for the design and synthesis of new materials. Indeed, biology forms these remarkable materials in aqueous environments under ambient conditions, and uses organic molecules – either as soluble additives or insoluble matrices – to control crystal growth. This MC-IIF research project investigated the use of bio-inspired approaches to synthesise porous inorganic nanoparticles of calcium carbonate and calcium phosphate. We then investigated the application of these particles to drug delivery. As compared with mesoporous silica, the calcium phosphate and calcium carbonate nanoparticles offer superior biocompatibility and biodegradation, together with acid-responsive solubility. These goals were encompassed under three principal objectives; (1) to synthesise porous calcium carbonate and calcium phosphate nanoparticles, (2) to understand the mechanisms of crystal nucleation and growth in nano-sized confinement, and (3) to develop the porous inorganic nanoparticles as drug delivery systems (DDS).
Description of Work Performed: A number of mesoporous solids were used as templates to control the formation of porous nanoparticles of calcium carbonate and calcium phosphate. These included nanoporous polymer capsules, mesoporous silica nanoparticles (MSNs) and mesoporous polymer nanoparticles. Crystal nucleation and growth within these templates was attempted using a range of bio-inspired methods such as use of polymer-induced liquid precursor (PILP) phases. We also developed a novel strategy employing combined nucleation and growth control, in which the MSNs were used as nucleating agents and fetuin as a growth inhibitor. Together, these two activities led to the formation of novel porous inorganic nanoparticles, hydroxyapatite (HA) and calcium carbonate. The potential activity of these particles for drug delivery was explored using bovine serum albumin (BSA) as a model compound. As an alternative strategy, the possibility of synthesising mesoporous calcium carbonate nanoparticles via an amorphous calcium carbonate (ACC) precursor phase was also investigated. Here, ACC nanoparticles were precipitated in ethanol/water solutions, where control over the size and stability of the ACC could be achieved according to the composition of the reaction mixture. Further addition of an organic phosphate was used to direct the formation of porous CaCO3 nanoparticles. Finally, a “nanocasting” strategy in which ACC particles were coated with a shell of paraffin before initiating crystallization was employed in order to maximise the porosity of the nanoparticles after transformation from ACC to crystalline polymorphs.
The two most successful methods were (1) the formation of mesoporous HA nanoparticles using controlled nucleation/ growth inhibition and (2) the synthesis of mesoporous calcium carbonate nanoparticles via the crystallisation of ACC nanoparticles in ethanol/ water in the presence of triethyl phosphate.
Summary of Principal Results Obtained: (1) MSNs were employed as seeds to promote the nucleation of HA, while fetuin, which is an abundant serum protein which regulates vascular calcification and bone metabolism, was used as a growth inhibitor. Together, these reagents acted to control the nucleation, aggregation and growth of HA, and resulted in flower-like mesoporous hydroxyapatite nanorods (MHANs) with controllable surface areas of 130–225 m2 g–1, particle sizes of 235–515 nm and tunable pore sizes of 21–31 nm. The particle sizes and pores sizes could be tuned according to the concentration of fetuin employed. The morphologies, porosities, compositions and structures of the MHANs were characterized using a range of techniques including Scanning and transmission electron microscopy, Surface area analysis, IR spectroscopy and X-ray diffraction. The mechanisms underlying the formation of the MHANs were also investigated by studying the effects of MSNs and fetuin on the nucleation and growth of the MHANs. These studies showed that fetuin – which is too large to enter the pores of the MSNs – can effectively inhibit HA growth, while the porous surface of the MSNs means that they can induce the nucleation of HA. Together, this generates a higher number density of smaller particles.
The synthesised MHANs were then investigated as potential drug delivery systems (DDS), where bovine serum albumin (BSA) was employed as a model, large-molecule drug. The results indicate that the MHANs exhibit a high payload to BSA (182 mg BSA per gram MHANs), which is significantly higher than solid HA nanorods (SHANs). This is due to the differences in the internal structures of these particles. The MHANs also exhibit a more sustained drug release profile than the SHANs, owing to their well-defined mesoporous structures. The resulting confinement of the drug limits diffusion, and provides an additional effect to general electrostatic adsorption. The intracellular uptake and cytotoxicity of the MHANs was also studied to evaluate their potential biological performance as drug carriers. This showed that the nano-sized MHANs are easily up-taken by the HeLa cells, and that they exhibit low cytotoxicity in the particle concentration range of 1.5 – 100 μg mL–1. These novel, porous inorganic nanoparticles therefore have potential for intracellular drug delivery.
(2) It is now recognised that many organisms precipitate crystalline biominerals such as calcium carbonate and calcium phosphate via amorphous precursor phases. This is thought to provide the organism with greater control over the crystallization process, and to give faster mineralisation rates. The use of amorphous precursor phases was therefore explored here as a way to generate mesoporous nanoparticles of calcium carbonate. ACC was precipitated from an ethanol/ water solution in the presence of triethyl phosphate (TEP) through exposure to ammonium carbonate vapour. Both the ethanol and phosphate additives control the size and stability of the ACC phase, enabling porous nanoparticles to be produced. This methodology resulted in the synthesis of novel mesoporous calcium carbonate nanoparticles (MCCNs). These exhibited a small and unique particle size ( 100 nm), a large mesopore size ( 20 nm) and a high surface area. The morphologies, porosities, compositions and structures of the MCCNs were characterized using a range of techniques. This technique was also elaborated to encapsulate the MCCNs within a shell of paraffin wax, and subsequent crystallization yielded hollow calcium carbonate nanoparticles. The potential of using these porous nanoparticles as drug delivery agents is currently under investigation.
Impact of the Project: This project developed novel, bio-inspired routes for the synthesis of porous inorganic nanoparticles, and then investigated the application of these structures in drug delivery and controlled release systems. This topic falls within the priority research areas of nanomedicine and more generally healthcare, which is of great significance to the EU, particularly in a time of an increasing elderly population. The research carried out within the Fellowship is therefore of immediate benefit to the EU by advancing a topic (targeted drug delivery) that could potentially benefit millions of people. Importantly, the methodologies and understanding developed within the project will also be valuable beyond an application of targeted drug delivery. The research will provide a novel route to the synthesis of porous inorganic nanoparticles under mild reaction conditions. This satisfies the increasing demand from the biomedical and chemical industries for nano-structured materials with specific mechanical, structural, degradable and biocompatible features and contributes to technological innovation through advancing water-based production methods effective at ambient temperatures. These provide an alternative to oil-derived polymers, thereby addressing current health- and energy-related challenges.
This Fellowship also had an impact on (i) the visiting researcher (Dr He), (ii) the European research groups with whom he collaborated (Meldrum and Sommerdijk), and (iii) the general public through outreach activities. The benefits to Dr He have included the experience of working in a different country with a different social environment and culture, improving his language skills, learning new research methods and the opportunity to attend international conferences. The research project undertaken also enabled Dr He to develop his interest and expertise in the fabrication of inorganic materials with controlled sizes and structures for bio-medical applications. Following a subsequent postodoctoral research fellowship at the National Institute of Health USA (Washington) Dr He will return to China to take up a position at a top, research-led Institute/University, where he will lead his own research group.
Dr He brought his extensive experience in the synthesis and application of mesoporous silica nanomaterials to both Meldrum’s and Sommerdijk’s groups, and they will thus be able to continue to profit from this in current/ future research programmes. Qianjun also brought unique experience in drug delivery and cell biology to Meldrum’s group, which has enabled the team to develop effective protocols for loading drugs into porous inorganic nanoparticles and to then investigate their controlled release. Importantly, the collaboration will be maintained beyond the Fellowship, where such strong links with a leading Chinese group will be valuable to the EU. With the economy and scientific community in China rapidly growing, it is of tremendous strategic importance for the EU to establish links with China, as this will inevitably provide the foundation for future economic and scientific benefits, and will allow from the EU to benefit from China’s growth and development. Finally, this Fellowship contributed to a range of outreach activities, aimed at transferring his knowledge to school children and the general public. Further information on this project can be found on the website http://qjhe.freeiz.com/News/MCF.htm(odnośnik otworzy się w nowym oknie).
Description of Work Performed: A number of mesoporous solids were used as templates to control the formation of porous nanoparticles of calcium carbonate and calcium phosphate. These included nanoporous polymer capsules, mesoporous silica nanoparticles (MSNs) and mesoporous polymer nanoparticles. Crystal nucleation and growth within these templates was attempted using a range of bio-inspired methods such as use of polymer-induced liquid precursor (PILP) phases. We also developed a novel strategy employing combined nucleation and growth control, in which the MSNs were used as nucleating agents and fetuin as a growth inhibitor. Together, these two activities led to the formation of novel porous inorganic nanoparticles, hydroxyapatite (HA) and calcium carbonate. The potential activity of these particles for drug delivery was explored using bovine serum albumin (BSA) as a model compound. As an alternative strategy, the possibility of synthesising mesoporous calcium carbonate nanoparticles via an amorphous calcium carbonate (ACC) precursor phase was also investigated. Here, ACC nanoparticles were precipitated in ethanol/water solutions, where control over the size and stability of the ACC could be achieved according to the composition of the reaction mixture. Further addition of an organic phosphate was used to direct the formation of porous CaCO3 nanoparticles. Finally, a “nanocasting” strategy in which ACC particles were coated with a shell of paraffin before initiating crystallization was employed in order to maximise the porosity of the nanoparticles after transformation from ACC to crystalline polymorphs.
The two most successful methods were (1) the formation of mesoporous HA nanoparticles using controlled nucleation/ growth inhibition and (2) the synthesis of mesoporous calcium carbonate nanoparticles via the crystallisation of ACC nanoparticles in ethanol/ water in the presence of triethyl phosphate.
Summary of Principal Results Obtained: (1) MSNs were employed as seeds to promote the nucleation of HA, while fetuin, which is an abundant serum protein which regulates vascular calcification and bone metabolism, was used as a growth inhibitor. Together, these reagents acted to control the nucleation, aggregation and growth of HA, and resulted in flower-like mesoporous hydroxyapatite nanorods (MHANs) with controllable surface areas of 130–225 m2 g–1, particle sizes of 235–515 nm and tunable pore sizes of 21–31 nm. The particle sizes and pores sizes could be tuned according to the concentration of fetuin employed. The morphologies, porosities, compositions and structures of the MHANs were characterized using a range of techniques including Scanning and transmission electron microscopy, Surface area analysis, IR spectroscopy and X-ray diffraction. The mechanisms underlying the formation of the MHANs were also investigated by studying the effects of MSNs and fetuin on the nucleation and growth of the MHANs. These studies showed that fetuin – which is too large to enter the pores of the MSNs – can effectively inhibit HA growth, while the porous surface of the MSNs means that they can induce the nucleation of HA. Together, this generates a higher number density of smaller particles.
The synthesised MHANs were then investigated as potential drug delivery systems (DDS), where bovine serum albumin (BSA) was employed as a model, large-molecule drug. The results indicate that the MHANs exhibit a high payload to BSA (182 mg BSA per gram MHANs), which is significantly higher than solid HA nanorods (SHANs). This is due to the differences in the internal structures of these particles. The MHANs also exhibit a more sustained drug release profile than the SHANs, owing to their well-defined mesoporous structures. The resulting confinement of the drug limits diffusion, and provides an additional effect to general electrostatic adsorption. The intracellular uptake and cytotoxicity of the MHANs was also studied to evaluate their potential biological performance as drug carriers. This showed that the nano-sized MHANs are easily up-taken by the HeLa cells, and that they exhibit low cytotoxicity in the particle concentration range of 1.5 – 100 μg mL–1. These novel, porous inorganic nanoparticles therefore have potential for intracellular drug delivery.
(2) It is now recognised that many organisms precipitate crystalline biominerals such as calcium carbonate and calcium phosphate via amorphous precursor phases. This is thought to provide the organism with greater control over the crystallization process, and to give faster mineralisation rates. The use of amorphous precursor phases was therefore explored here as a way to generate mesoporous nanoparticles of calcium carbonate. ACC was precipitated from an ethanol/ water solution in the presence of triethyl phosphate (TEP) through exposure to ammonium carbonate vapour. Both the ethanol and phosphate additives control the size and stability of the ACC phase, enabling porous nanoparticles to be produced. This methodology resulted in the synthesis of novel mesoporous calcium carbonate nanoparticles (MCCNs). These exhibited a small and unique particle size ( 100 nm), a large mesopore size ( 20 nm) and a high surface area. The morphologies, porosities, compositions and structures of the MCCNs were characterized using a range of techniques. This technique was also elaborated to encapsulate the MCCNs within a shell of paraffin wax, and subsequent crystallization yielded hollow calcium carbonate nanoparticles. The potential of using these porous nanoparticles as drug delivery agents is currently under investigation.
Impact of the Project: This project developed novel, bio-inspired routes for the synthesis of porous inorganic nanoparticles, and then investigated the application of these structures in drug delivery and controlled release systems. This topic falls within the priority research areas of nanomedicine and more generally healthcare, which is of great significance to the EU, particularly in a time of an increasing elderly population. The research carried out within the Fellowship is therefore of immediate benefit to the EU by advancing a topic (targeted drug delivery) that could potentially benefit millions of people. Importantly, the methodologies and understanding developed within the project will also be valuable beyond an application of targeted drug delivery. The research will provide a novel route to the synthesis of porous inorganic nanoparticles under mild reaction conditions. This satisfies the increasing demand from the biomedical and chemical industries for nano-structured materials with specific mechanical, structural, degradable and biocompatible features and contributes to technological innovation through advancing water-based production methods effective at ambient temperatures. These provide an alternative to oil-derived polymers, thereby addressing current health- and energy-related challenges.
This Fellowship also had an impact on (i) the visiting researcher (Dr He), (ii) the European research groups with whom he collaborated (Meldrum and Sommerdijk), and (iii) the general public through outreach activities. The benefits to Dr He have included the experience of working in a different country with a different social environment and culture, improving his language skills, learning new research methods and the opportunity to attend international conferences. The research project undertaken also enabled Dr He to develop his interest and expertise in the fabrication of inorganic materials with controlled sizes and structures for bio-medical applications. Following a subsequent postodoctoral research fellowship at the National Institute of Health USA (Washington) Dr He will return to China to take up a position at a top, research-led Institute/University, where he will lead his own research group.
Dr He brought his extensive experience in the synthesis and application of mesoporous silica nanomaterials to both Meldrum’s and Sommerdijk’s groups, and they will thus be able to continue to profit from this in current/ future research programmes. Qianjun also brought unique experience in drug delivery and cell biology to Meldrum’s group, which has enabled the team to develop effective protocols for loading drugs into porous inorganic nanoparticles and to then investigate their controlled release. Importantly, the collaboration will be maintained beyond the Fellowship, where such strong links with a leading Chinese group will be valuable to the EU. With the economy and scientific community in China rapidly growing, it is of tremendous strategic importance for the EU to establish links with China, as this will inevitably provide the foundation for future economic and scientific benefits, and will allow from the EU to benefit from China’s growth and development. Finally, this Fellowship contributed to a range of outreach activities, aimed at transferring his knowledge to school children and the general public. Further information on this project can be found on the website http://qjhe.freeiz.com/News/MCF.htm(odnośnik otworzy się w nowym oknie).