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Development of Irinotecan-Loaded PCL Nanoparticles and Their Cytotoxicity against Glioma Cells

Development and Optimization of Irinotecan-Loaded PCL Nanoparticles and Their Cytotoxicity against Primary High-Grade Glioma Cells


High-grade gliomas (HGGs) are highly malignant tumors with a poor survival rate. The inability of free drugs to cross the blood–brain barrier and their off-target accumulation results in dose-limiting side effects. This study aimed at enhancing the encapsulation efficiency (EE) of irinotecan hydrochloride trihydrate (IRH) within polycaprolactone (PCL) nanoparticles with optimized size and charge. Materials and Methods: IRH-loaded PCL nanoparticles were formulated using either the single emulsion (O/W, W/O, and O/O) or double emulsion (W/O/O and W/O/W) solvent evaporation techniques. The nanoparticles were characterized for their size, zeta potential, and EE, with the optimized nanoparticles being characterized for their drug release and cytotoxicity. Results: The amorphization of PCL and the addition of electrolytes to the aqueous phases of the W/O/W emulsion produced spherical nanoparticles with a mean diameter of 202.1 ± 2.0 nm and an EE of 65.0%. The IRH-loaded nanoparticles exhibited zero-order release and were cytotoxic against primary HGG cells. Conclusion: The amorphization of PCL improves its EE of hydrophilic drugs, while the addition of electrolytes to the aqueous phases of the W/O/W emulsion enhances their EE further. IRH-loaded PCL nanoparticles have the potential to deliver cytotoxic levels of IRH over a sustained period of time, enhancing the cell death of HGGs.


Malignant brain tumors are associated with high mortality and morbidity rates, owing to the lack of long-term disease control. The majority of malignant brain tumors that affect adults are gliomas, with high-grade gliomas (HGGs) accounting for 75%. For each 100,000 population, 3 to 5 persons develop gliomas annually, with a higher frequency in men. Although the fifth and sixth decades of life are the most prominent for glioma incidence, gliomas can develop at any age. In 2016, the WHO included genetic criteria in classifying gliomas, which resulted in identifying anaplastic astrocytoma, anaplastic oligodendroglioma, and mixed anaplastic oligoastrocytoma (grade III) and glioblastoma (grade IV) as high-grade gliomas, accounting for the majority of cases, with a 60 to 70% incidence rate. Several hurdles limit the efficacy of glioma treatment: glioma cells can metastasize to other tissues in the brain and their irregular margins make complete resection difficult; the blood-brain barrier (BBB) prevents drug molecules from entering the brain at therapeutic concentrations; cancer stem cells contribute to tumor initiation and therapeutic resistance and glioma cells are immortalized in nature and any cells left behind during surgery will develop into a new tumor. The standard treatment for gliomas is the Stupp protocol, in which patients undergo surgical resection, followed by radiotherapy and subsequent treatment with temozolomide.



Solubility (A) and stability of IRH in both dH20 (B) and PBS (C) over 16 days at 8, 25 and 37 ◦C


This treatment protocol has been shown to increase survival to 14.6 months, compared to 12.1 months with radiotherapy alone. Other treatment options include Gliadel® wafers made of biodegradable poly-carboxy phenoxy propane containing the chemotherapeutic drug Carmustine (Bis-chloroethyl nitrosourea). However, a Cochrane report demonstrated that the Gliadel wafers provided no significant clinical benefit for newly diagnosed HGG patients, with a small but significant clinical benefit to recurrent HGG patients. Furthermore, the Gliadel wafers suffer from poor drug penetration into the brain parenchyma, which means that the drug does not reach the deep-seated tumor tissue, and reoperation is needed to implant additional wafers once they have finished releasing the drug, which introduces further invasiveness to the patient. The problem with alkylating agents such as temozolomide is the presence of methyl guanine methyltransferase (MGMT), which can counteract their mechanism of action, leading to multidrug resistance (MDR). Therefore, there has been some research into the second line of drugs such as Carboplatin, Oxaliplatin, and Irinotecan hydrochloride trihydrate (IRH). However, the systemic delivery of these chemotherapeutic drugs results in serious unwanted side effects for the patient, such as vomiting, diarrhea, hair loss, neutropenia, myelosuppression, and pain. Traditional chemotherapeutic drugs also have poor in vivo stability, with off-target tissue accumulation, low bioavailability, and an inability to cross the BBB, leading to larger doses being administered in order to achieve the therapeutic concentrations needed in the brain, resulting in the deterioration of the patient’s overall health. Encapsulating drugs within nanoparticles (NPs) can offer a wide range of benefits that overcome some of the issues of traditional chemotherapy and could help to improve cancer. Drug-loaded NPs can cross the BBB by passive diffusion and accumulate in the tumor via the enhanced permeability and retention (EPR) effect, where they release their drug in a sustained fashion by diffusion. NPs can protect drugs against early metabolism and reduce off-target tissue accumulation, which in turn increases treatment efficacy and protects healthy tissues. Drug encapsulation can extend the half-life of drugs and reduce the frequency of administration. Targeting NPs by surface coating of antigen-specific antibodies can enhance the endocytosis uptake of NPs, where drugs encapsulated within NPs can escape multidrug resistance regulated by p-glycoprotein transporters. Among NPs, biodegradable polymers of synthetic or natural origin are widely favored for drug loading and delivery, since they metabolize and require no surgical removal after drug release. Synthetic polymers are utilized in drug delivery due to their uniformity and ease of manufacture as well as scale-up. Unlike phospholipids, synthetic polymers are inert materials with no oxidative by-products released upon degradation and they possess versatile properties that can be manipulated to suit their intended use.




Several synthetic polymers have been shown to offer advantages in drug delivery, such as poly (lactic acid) (PLA), polycaprolactone (PCL), poly (butyl-cyanoacrylate) (PBCA), poly (glycolic acid) (PGA), poly (lactic-co-glycolic acid) (PLGA) and poly (amino acids) . The choice of polymer depends on the final application of the NPs and their behavior can be affected by their crystalline or amorphous nature, porosity, biocompatibility and degradation profile. Depending on their chemistry, synthetic polymers tend to undergo swelling followed by erosion or degradation, with drug release taking place by both diffusion, during the early stages of release, and degradation, during the later stages of release. Several techniques are used to manufacture drug-loaded polymeric NPs, such as solvent evaporation, nanoprecipitation, emulsification/solvent diffusion, salting out and supercritical fluid (SCF) technology. Drugs can be formulated into NPs as either nanospheres or nanocapsules. Nanospheres are compact spheres where the drug is either dissolved or dispersed in a polymer matrix, with dissolved drugs forming homogeneous systems, while dispersed drugs tend to form heterogeneous systems. With nanocapsules, the drug is surrounded by a polymeric coat. Drugs can either be loaded onto pre-manufactured NPs by covalent or non-covalent interactions or loaded into the NPs during their manufacture.



The impact of sonication time (A), polymer amount (B), drug amount (C), sonication amplitude (D), surfactant type (E), pH and PVA concentration (F) on the average particle size, PDI and zeta potential of the nanoparticles manufactured using the single emulsion technique.


Hydrophilic drugs are difficult to formulate and encapsulate into the matrix of a hydrophobic polymer. This is because they have a poor affinity towards hydrophobic polymers and they favor the water phase during the emulsification process, which results in poor encapsulation efficiency (EE). In addition, the large surface area of the NPs can lead to premature drug release due to a large amount of the drug being on the surface of NPs, instead of being encapsulated on the inside. PCL is a semi-crystalline and slow degrading polymer, which can alleviate the issue of rapid drug release. However, there are no publications on manipulating PCL to increase its efficiency in encapsulating hydrophilic drugs. PCL degradation is slower than PLGA, which enhances its application in extending drug release for periods exceeding one year. Moreover, it has the advantages of high permeability to small drug molecules.


A study had previously reported sustained release of proteins using PCL. Other advantages of PCL that encourage its use over PLGA are: PCL is considered more economic than PLGA, it does not induce an acidic environment upon degradation, and it provides sustained release and biodegradation over longer periods of time, which would, in turn, have less impact on homeostasis.


Their preliminary studies demonstrate that the solvent evaporation technique is efficient in producing spherical and monodisperse PCL NPs with reasonable stability. Therefore, they selected this technique to develop PCL NPs with tailored mechanical properties to enable a high encapsulation efficiency of IRH. IRH is potent hydrophilic chemotherapy that inhibits topoisomerase, leading to the inhibition of DNA replication. The mechanism of action of IRH is not limited by MGMT methylation. Additionally, IRH and its highly potent metabolite (SN-38) are not substrates of the p-glycoprotein transporter and therefore will not face the challenge of MDR. However, IRH can face difficulty crossing the BBB by passive diffusion because its molecular weight is 677 Da, which is above the limit (160 Da) for a hydrophilic drug. Moreover, IRH undergoes hydrolysis at physiological pH (7.4), which converts the active lactone form (stable below pH 5) to its less active carboxylate form (stable above pH 8). Therefore, it is necessary to protect the active form of the drug by shielding it from the physiological conditions, until reaching the acidic environment of the tumor. This study will investigate the influence of different parameters such as drug and polymer concentration; pH; type, molecular weight and concentration of surfactants; PCL amorphization and use of electrolytes on the size, polydispersity index (PDI), surface charge and encapsulation efficiency. The release profile and cytotoxicity will be evaluated for the optimized IRH-loaded PCL NPs.