Intrinsically green fluorescent PAMAM as nanocarrier and nanoprobe for traceable and controlled drug delivery and bio-imaging
Creators: name: Wang, Guoying
Publisher: name: Macquarie University
Year: 2023
Subjects: Other education not elsewhere classified

description: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegeneration disease that mainly affects motor neurons that control muscle movement. Patients normally die with respiratory muscle failure within 3~5 years from the time of diagnosis. Current therapeutic approaches can only modestly slow the disease progress. Recent advances in nanotechnology have shown great potential for developing a nano-based therapeutic strategy in enhancing the power of ALS therapeutic agents. Those benefits including but not limited to: (1) Improve the drug's bioavailability/bio-stability; (2) Enhance the biological barriers penetration; (3) Target specific disease sites; (4) Traceable drug/gene delivery. Therefore, it is of great importance to develop multifunctional nanomaterials that can be used in delivering ALS treatment drugs. Among numerous nanomaterials, intrinsically fluorescent Poly(amidoamine) dendrimers (IF-PAMAM) have attracted extensive attention in the fields of biological imaging and drug delivery due to their detectable signals, small size, excellent biocompatibility, abundant functional terminal groups, and superior photo-stability. To date, several intrinsically blue fluorescent PAMAM dendrimers have been successfully applied for traceable drug/gene delivery. However, those blue fluorescent dendrimers typically require UV as the excitation light, which may cause side effects to healthy cells or tissues. Additionally, under UV excitation, the emission spectra from those PAMAM dendrimers were mainly concentrated at the range of 400 ~500 nm, which was close to the spontaneous fluorescence spectra from biological tissues, thus affecting their effect as fluorescent probes for <em>in vivo </em>application. Therefore, the development of biocompatible, photo-stable, and high fluorescence performance PAMAM dendrimers for traceable drug delivery or bio-imaging is still a challenge. In my PhD project, we firstly developed a novel highly biocompatible and label-free (intrinsically) fluorescent PAMAM through a simple reaction of NH<sub>2</sub>- terminated PAMAM (Generation 5) with acetaldehyde. This unique intrinsically fluorescent PAMAM (FG5) emitted a strong green fluorescence under the maximum excitation around 485 nm. Due to the replacement of the positively charged amino groups with neutrally charged methyl groups, the inherent cytotoxicity of NH<sub>2</sub>-terminated PAMAM was largely eliminated. The higher excitation wavelength made the FG5 a much safer drug carrier for tracking and bio-imaging. The fluorescent anti-cancer drugs Doxorubicin (DOX) were selected as model drugs to evaluate the dual-tracking (nanocarrier and payload) capability of this system, thus providing base for delivering other clinic important drugs. The effective delivery of nanocomposites to melanoma cancer cells (SKMEL28) and excellent intracellular tracing of these nanoparticles suggest that these novel FG5 are warrant for further study of other clinical drugs. Selective targeting of motor neuron protective drugs to the central nervous system (CNS) remains a great challenge for effective amyotrophic lateral sclerosis (ALS) therapy due to the presence of blood-brain barrier (BBB) and blood spinal cord barrier (BSCB). Therefore, the second aim of the thesis was to assess the CNS targeting capability of the fabricated FG5. In this chapter, Transferrin (Tf) was selected as a transport ligand to facilitate the BBB penetration of carrier/drug nanocomplexes. Neuron protection drug Edaravone (EDV) was encapsulated in the PEGylated FG5 (FGP), and Tf functionalized FG5 (FGP-Tf) by a solvent replacement (precipitation) method. The enhanced BBB transportation and elevated neuron protection function of EDV loaded FGP-Tf (FGP-Tf/EDV) were confirmed by the <em>in vitro </em>BBB model and motor neuron cells model. To the best of our knowledge, this is the first report of IF-PAMAM as a nanocarrier for neuroprotective drug delivery, which is of great importance in developing IF-PAMAM based nanomedicines for ALS treatment. To accelerate the transformation of IF-PAMAM-based nanomedicines from theoretical to clinical, it is practically required to evaluate the <em>in vivo </em>behaviour through standard animal models. Therefore, the third aim of this thesis was to fill the gaps in utilizing intrinsically fluorescent PAMAM dendrimers in <em>in vivo </em>models. The excellent performance for <em>in vivo </em>imaging and real-time tracking of our fabricated FG5 was verified by the strong <em>in vivo </em>green fluorescent signals, superior photo-stability, and very low fluorescent background (noise) from biological organisms. Additionally, we also provided a simple and effective zebrafish platform to evaluate the <em>in vivo </em>behaviour of IF-PAMAM based nanoprobes, which was beneficial for optimizing IF-PAMAM based nanostructure for future <em>in vivo </em>drug delivery. Through the comprehensive study in this thesis, we thereby developed a novel intrinsically green fluorescent PAMAM with excellent biocompatibility, remarkable fluorescent performance, superior photo-stability that provided the fundamental nanomaterial (multifunctional PAMAM) for this thesis. Importantly, the enhanced BBB transportation and neuron protection function of FGP-Tf/EDV in <em>in vitro </em>models, accompanied with the prolonged blood circulation, and excellent tissue diffusion function of FGP-Tf in <em>in vivo </em>zebrafish model provided a solid basis for further evaluating the therapeutic effect of FGP-Tf/EDV in ALS animal models.