Acute Myeloid Leukemia (AML) is a type of blood malignancy with thousands of new cases yearly in Europe[1], [2], tens of thousands in the USA[3], and approximately 120 thousands new cases globally every year[4]. Although therapies using Tyrosine Kinase Inhibitors (TKIs) for AML have been studied more and more intensely in the last 20 years (over 3400 research papers found in the WoS database), the standard of care is still a combination of cytosine arabinoside (ara-C) with an anthracycline, since almost 5 decades ago[5]. Still, it seems that in the near-future the medical world might establish the real impact of TKI-based AML therapy, since there are already many ongoing clinical trials focused on this aspect[6]. Nevertheless, due to their more acceptable safety profile with less off-target side effects, TKIs hold a remarkable advantage that makes them suitable and promising for combinatorial or sequential treatment regimens together with other small molecule inhibitors or traditional “broad spectrum” chemotherapeutics[6].
Tyrosine Kinase Inhibitors are a class of therapeutic molecules capable of inhibiting the kinase activity of different tyrosine kinases[7], functional proteins having a key role in intracellular signal transduction, and involved in a multitude of biological pathways concerning cell regulation, proliferation, differentiation etc[8], [9]. FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase receptor known as overexpressed in 80% of AML cases, and mutated in 30%[10]. Thus, it is a highly significant target for TKI-based AML therapy and multiple TKIs have been investigated for this purpose, such as Sunitinib, Sorafenib, Lestaurtinib, Midostaurin, Crenolanib, and Quizartinib[11]–[13]. Among these, Midostaurin (MDS) was FDA approved in 2017 for newly diagnosed AML patients[14] and has shown improvement in overall survival and event-free survival of patients if used in combination with standard chemotherapy, without increasing the incidence of severe adverse events[15], [16]. Regardless, TKIs are hydrophobic molecules with low bioavailability, and chemotherapeutics are eminently toxic and induce unwanted side-effects. Considering these, efforts are still required in order to reduce their impact onto the quality of life of the patients, via approaches that can improve drug loading, targeting, focused release, and thus the overall clinical outcome.
Nanotechnology can offer certain improvements in these regards, in the form of nano-pharmacology[17], [18], by combining well-known and studied therapeutic drugs with nanoparticles (NPs) and the new and compelling properties rendered by them. The high surface-to-volume ratio of NPs makes them convenient drug carriers with a high capacity for loading cargo, and their chemistry allows ease of further functionalization with molecules such as polymers. Furthermore, based on the chemical properties of the polymeric shell arise a multitude of advantages, such as the possibility for grafting targeting moieties, the ability to encapsulate therapeutic molecules or contrast agents of various chemistries as payload, and the wherewithal for controlled release of the payload at the target site based on slight endogenous differences such as pH, ionic strength, temperature, or the concentration of other chemical components[19], [20]. Moreover, the composition of the NP core offers some exceptional physical and optical properties to the compound such as the light scattering and field enhancement effects in the case of noble-metal nanoparticles or the heat-releasing capabilities in the magnetic ones[21]. Multiple applications can arise from these properties among which contrast agents for imaging or tools for theranostics purposes are most notable[22]–[24].
Stimuli-responsive polymers can aid greatly in the aspect of reducing some of the major limitations of chemotherapeutics such as ubiquitous biodistribution, non-specificity, and subsequent systemic toxicity, as they can ensure bioavailability and release of the therapeutic molecules chiefly at the diseased site, allowing lowering of the dose and dosage frequency while maintaining targeted toxicity [20], [25]. There are two main strategies for pH-sensitive drug release: one is based on acid-labile linkers between the drug and the rest of the particle allowing the prodrug to become active after hydrolysis, and one is based on ionizable polyelectrolytes that dramatically change their conformation as a function of the environmental pH[20]. Moreover, the polymers that contain hydrophobic or amphiphilic sections can improve the solubility, bioavailability and transportation of hydrophobic drugs such as TKIs, functioning as stealth vectors, as they can engulf the cargo in lipophilic pockets within their structure, and deliver them throughout the body with less clearance.
There are some research papers focused on pH-sensitive polymers and pH-triggered drug release, such as that published by Chen et al[26] where algae-based carrageenan oligosaccharides (CAOs) were used as a green reducing agent for GNP synthesis and as a pH-triggered delivery system for epirubicin (EPI), with negligible release under physiological pH, in hepatic and epithelial cells. In another study, the hydrophobic poly (beta-amino ester) core of polymeric pH-sensitive STEALTH® nanoparticles containing the lipophilic Paclitaxel (PTX) undergo rapid dissolution at pH below 6.5 and drug release, showing good toxicity in Jurkat acute lymphoblastic leukemia cells compared to the free drug[27]. Also, anthracyclines such as doxorubicin (DOX) and idarubicin (IDA) were loaded, based on both hydrophobic and electrostatic interactions, and delivered to leukemia cells via oxidized phospholipid-based pH-sensitive micelles that show an accelerated drug release behavior under acidic conditions (pH 6) compared to 7.4[28]. More, DOX was co-loaded with a short chain ceramide (C6-ceramide) in a PEGylated bioactive lipids-based micelle system for a synergistic cytotoxic effect on drug resistant leukemia cells, resulting in improved IC50 values and apoptosis, as a result of controlled drug release at pH values of 6 and 5[29]. Unimolecular micelles composed of cyclodextrin-{poly(lactic acid)-poly(2-(dimethylamino) ethyl methacrylate)-poly[oligo(2-ethyl-2-oxazoline)methacrylate]}21 [CD-(PLA-PDMAEMA-PEtOxMA)21] were used for DOX encapsulation in the PDMAEMA and PLA layers, as well as in situ formation spots for GNP synthesis, in a study by Lin et al[30], and drug release was shown to be enhanced with the lowering of the pH, from 7.4 to 6.5 and 5, respectively. More, an amphiphilic copolymer of PCL-PDMAEMA linked by a disulfide bond was used to efficiently deliver DOX to HepG2 and MCF-7 cells based on a pH-induced polymer re-conformation and the GSH reduction of the S-S bond[31]. In a recent work, hydrophobic PTX was encapsulated in a FFACD peptide-based hydrogel and its’ toxicity against K562 leukemia cells was confirmed. If the hydrogel was synthesized in the presence of amino-acid Arginine, it showed a highly improved PTX release at pH 6 compared to pH 7.4[32]. Also, the pH-sensitive amphiphilic polymer poly [(1,4-butanediol)-diacrylate-β-NN-diisopropylethylenediamine]-polyethyleneimine (BDP) was synthesized by Yin et al[33] and loaded with PTX leading to improvements in drug loading, murine breast cancer cell toxicity and mice survival. This system proved stable at neutral and tumor extracellular pH, but the intra-endo/lysosomal acidic environment induced a strong controlled drug release.
On the other hand, very few works have been published to study the pH-controlled release of hydrophobic drugs such as TKIs, from amphiphilic nano-pharmacological systems, and even less are focused in hematological malignancies, as seen discussed below. Sarkar et al[34] have developed poly(ethylene glycol)-block-poly(propyleneglycol)-block-poly(ethylene glycol) (PEG − PPG − PEG) coated GNPs where the hydrophobic PPG functions as a reducing agent for the GNP synthesis, and an excellent carrier for the ZD6474 epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR)-specific TKI. The obtained particles show stability at physiological pH and a sustained release profile at an acidic pH of 5.2, as well as promising in vitro and in vivo anti breast-cancer results. Hong et al[35] used lipid polymeric nanoparticles conjugated with dual pH‑responsive and targeting peptides for the delivery of Afatinib, an anti-EGFR TKI into colorectal adenocarcinoma (CRC) cells. The drug was loaded in the polylactic-co-glycolic acid (PLGA) core, which was surrounded by a layer of PEG-lipids, further modified with pH sensitive and specific cell-penetrating peptides, showing increased drug release at pH 6.5 and modulated apoptosis, drug resistance, and metastasis. Focused on leukemias, recently, Erdagi and Yildiz[36] obtained polymeric nanoparticles with a methoxy poly (ethylene glycol) (mPEG) shell and a poly (ε-caprolactone) (PCL) hydrophobic core where Imatinib was encapsulated. They showed pH-sensitive release comparing buffer solutions of pH 7.4 and 3.5, and an improved cytotoxicity effect on K562 leukemia cells, compared to the free drug. Imatinib was also used by Cortese et al[37] in a chitosan and PCL nanoparticle system, where a much quicker drug release was observed at pH 4 and 6 compared to 7.4, and a cytotoxic effect via apoptosis was induced in KU812 chronic myeloid leukemia cells. The same group then published research on an even more complex, pH-triggered, dual TKI delivery system[38], with a synergistic cytotoxic effect on KU812 cells. The authors developed a pH-sensitive chitosan core loaded with Nilotinib, surrounded by a PCL shell incorporated with a mixture of sodium bicarbonate and potassium tartrate (NaHCO3 + KC4H5O6) which quickly generate CO2 bubbles in acidic conditions, leading to the release of the Nilotinib. On top of this, there is another shell layer of protease-sensitive dextran loaded with Imatinib. Thus, once incorporated, there is a chain of reactions that release the TKIs from the nanoparticles into the cells and induce cell death.
In our previous work by Simon et al[12], a pioneering study regarding the efficient loading of gold nanoparticles (GNPs) with selected FLT3 inhibitors, we showed that Pluronic capped, Midostaurin (MDS)-conjugated GNPs might be promising anti-leukemic candidates with a high drug loading value of 80%, a drug release rate of 50% in simulated lysosomal conditions after 24 hours, a clear reduction of cancer cell proliferation compared to control, and a superior anti-cancer effect compared to the free drug. Another work reported the use of a nanoparticle-complex based on different FLT3 inhibitors, namely Midostaurin, Sorafenib, Lestaurtinib, and Quizartinib, which were independently loaded onto gelatin-coated gold nanoparticles with promising results against THP1 acute myeloid leukemia cells[39]. We also reported that such nanobioconjugates are superior when compared with the drug alone, with data confirmed by state-of-the-art analyses of internalization, cell biology, gene analysis for FLT3-IDT gene, and Western blotting to assess degradation of the FLT3 protein[5]. Herein, we further our research by studying the MDS loading and controlled release behavior when conjugated onto GNPs functionalized with polymers that, in specific conditions, can have stimuli responsive properties as is the case of Polyvinylpyrrolidone (PVP). Hence, an optimization of the compound targetability can be achieved. Indeed, our results show that drug loading was attained to a higher degree due to the hydrophobicity of the drug molecule and its’ propensity to shield within the polymer. When studied in conditions that simulate lysosomal entrapment (acidic pH and presence of glutathione) as well as in control conditions simulating the extracellular fluids (physiological pH and ionic strength) over a time span of 24 hours, the PVP-capped nanoparticles demonstrated improved drug delivery within the target site in comparison with PLU-coated nanoparticles due to the controlled release effect, induced by the acidic pH. The cytotoxicity of the MDS-loaded GNPs was compared to that of the free drug molecule, with very effective results on the MV-4-11 (CRL-9591™) target cell line (cell viability below 15%) that carries the FLT3-ITD mutation [40], [41] and little observable effect in the case of negative control, OCI-AML-3 (ACC 582) cell line. Moreover, the internalization and distribution of the particle-based compounds inside cells and cellular compartments can be assessed using dark field microscopy imaging. This feature is of great importance when considering the efficient administration and the targetability of the drug cargo.