Effects of Organic Acids on Drug Release from Ternary Polypeptide Nanoparticles Entrapping Carfilzomib

Abstract

Carfilzomib (CFZ) is an FDA-approved proteasome inhibitor with antineoplastic properties against various cancers, yet its short blood retention time after intravenous injection (< 30 min) makes clinical applications limited to multiple myeloma. We previously developed ternary polypeptide nanoparticles (tPNPs) as a new nanoparticle formulation of CFZ to overcome these limitations. The formulation was prepared by polyion complexation between poly(ethylene glycol)-poly(L-glutamate) block copolymers (PEG-PLE) and CFZ-cyclodextrin (CD) inclusion complexes, where CDs were positively charged with 7 primary amines attached while PEG-PLE carried 100 carboxyl groups per polymer chain. Although tPNPs greatly improved biostability of CFZ, CFZ-loaded tPNPs (CFZ-tPNPs) still showed burst drug release and mediocre drug retention under physiological conditions. To address these issues, organic acids are tested as stabilizers in this study to improve particle stability and drug retention for tPNPs. Charge densities in the core of CFZ-tPNPs were optimized with selected organic acids such as citric acid (CA) and lactic acid (LA) at varying mixing ratios. Organic acids successfully maintained small particle size suitable for intravenous injection and drug delivery (diameters < 60 nm), improved CFZ solubility (> 1 mg/mL), allowed for lyophilization and easy reconstitution in various buffers, enhanced drug retention (> 60% post 24 h incubation), and suppressed burst drug release in the first 6 h following solubilization. These results demonstrate that organic acid stabilized tPNPs are useful as an injection formulation of CFZ, which may expand the utility of the proteasome inhibitor.

INTRODUCTION

Carfilzomib (CFZ) is a second generation proteasome inhibitor approved by the FDA in 2012 for the treatment of relapsed or refractory multiple myeloma^1–2. The tetrapeptide epoxyketone induces apoptosis by inhibiting proteasomes that otherwise would degrade many intracellular proteins in cancer cells for survival³. The FDA-approved proteasome inhibitor is used as a formulation (known as Kyprolis^®), which includes CFZ as an active pharmaceutical ingredient along with other chemical substances such as sulfobutylether beta-cyclodextrin, citric acid, and sodium hydroxide for pH adjustment⁴. The cyclodextrin (CD) in Kyprolis plays an important role in improving aqueous solubility of hydrophobic CFZ by forming inclusion complexes with the drug because carfilzomib is practically insoluble in aqueous solutions (10 μM in PBS)⁵. However, the current CFZ formulation requires 50 times more CD to dissolve CFZ (typically 3,000 mg of CD is needed for 60 mg of CFZ), which poses a major limitation of CD-based CFZ formulations causing toxicity at high concentrations. The CD-CFZ formulations also have difficulties in controlling the release of CFZ, which is significant to achieve safe and effective chemotherapy using the proteasome inhibitor in the clinic⁶.

To address these issues, we previously developed ternary polypeptide nanoparticles (tPNPs) as a new injection formulation for CFZ⁷. tPNPs are prepared by entrapping inclusion complexes of CFZ and heptakis(6-amino-6-deoxy)-β-cyclodextrin(hepta-hydrochloride) (HaβCD) in a self-assembling polyion complexes made of poly(ethylene glycol)-conjugated anionic polypeptides such as polyglutamate (Figure 1). CFZ-loaded tPNPs (CFZ-tPNPs) improved entrapment and aqueous solubility of CFZ while extending proteasome inhibition in cancer cells for greater efficacy than free drug and current CD-based formulations⁷. Despite these exciting results, CFZ-tPNPs still need further improvement in suppressing initial burst drug release and extending drug release half-life in an early stage of treatment (approximately 3 h). One possible solution to these problems is to stabilize the core of tPNPs without hampering drug release from the nanoparticles for fast yet prolonged drug responses. We hypothesized that organic acids stabilize polyionic complexes of HaβCD and tPNPs and extend drug release half-life for CFZ while suppressing burst drug release. This study focuses on determining the effects of organic acids commonly used in drug formulation design, such as citric acid (CA) and lactic acid (LA), on drug entrapment, drug release profiles, and overall particle stability of CFZ-tPNPs.

Figure 1.

Preparation of organic acid stabilized tPNPs for improved particle stability and drug retention

We aimed the minimum drug entrapment to be greater than 10 percentage by weight (wt%) for an active pharmaceutical ingredient (i.e. CFZ) with respect to other ingredients in the drug formulation (polymer, CD, and organic acids), which is 5 times greater than the current FDA-approved injection formulation of CFZ⁴. CFZ is a proteasome inhibitor that selectively causes cytotoxic effects on targeted cancer cells, and the drug is less likely to damage non-cancer cells that have low response to proteasome inhibition compared to proliferation cancer cells^8–10. Thus, we anticipate that high drug entrapment of CFZ-tPNPs is beneficial to increase drug accumulation in targeted sites in the body such as tumor tissues with reduced toxicity to patients.

In this study, drug release profiles were analyzed to determine drug release half-life (t_1/2) in the initial stage and later stage (t_{1/2, fast} and t_{1/2, slow}), which provide information on burst and sustained drug release, respectively. Small molecule drugs are generally cleared from the body quickly through renal filtration or hepatic degradation. The blood retention half-life of CFZ is between 4–39 minutes when injected intravenously using a CD formulation⁶. Our previous study showed that tPNPs extend t_{1/2, fast} of CFZ up to 3 h, and thus tPNPs are expected to extend the blood retention half-life and duration of action for CFZ by protecting the proteasome inhibitor in the body. We expect that tPNPs stabilized by organic acids may further suppress the drug fast release half-life due to improved particle stability. CA and LA were selected as organic acids for testing the half-life in simulation because the toxicity profiles of these acids as excipients are reported minimal^11–14. Particle stability is assessed in pH-controlled buffer solutions mimicking in vivo conditions by measuring particle size before and after freeze drying of drug-loaded nanoparticles. We also monitored the particle stability in the presence and absence of drug to better investigate interactions between organic acids and drugs inside the nanoparticle. We determined particle stability after lyophilization of CFZ-tPNPs because freeze drying is important for long storage of drug-loaded nanoparticles for clinical applications^15–16. Freeze-dried CFZ-tPNPs were then reconstituted in a buffer solution to determine drug concentrations. All data was obtained from triplicate experiments and reported as mean values with standard deviations.

MATERIALS AND METHOD

Materials

Carfilzomib (free base, >99%) was purchased from LC Laboratories (Woburn, MA). Poly(ethylene glycol)-block-poly(L-glutamic acid sodium salt) with 5 kDa PEG and 100 repeating units of glutamate (PEG-PLE) was purchased from Alamanda Polymers (Huntsville, AL). Heptakis (6-deoxy-6-amino)-beta-cyclodextrin heptahydrochloride (HaβCD) was purchased from Cyclolab R&D Laboratory Ltd. (Budapest, Hungary). Citric acid was purchased from EM Science (Gibbstown, NJ). LA was purchased from Spectrum (Gardena, CA). Slide-A-Lyzer MINI Dialysis Units 20,000 MWCO and HPLC grade water were purchased from Thermo Fisher Scientific (Rockford, IL).

Nanoparticle preparation

Nanoparticles were prepared by a modified nanoprecipitation method. For empty tPNPs, the starting procedure was as follows. HaβCD (4 mg) and PEG-PLE (2 mg) both dissolved in deionized water at a concentration of 10 mg/mL were mixed in a 1.5 mL conical tube. The resulting mixture was sonicated in a bath sonicator to form drug free tPNPs. For drug-loaded nanoparticles, 5 different CFZ/tPNPs were prepared following the method we previously reported⁷. Briefly, CFZ and HaβCD were dissolved in ethanol and water respectively, and the solutions were mixed in a 50 mL round bottom flask to form inclusion complexes at a drug to cyclodextrin mass ratio of 1:4. Organic acids (CA and LA) were added to the flask to stabilize the complexes. The mixing ratio of CA and LA was determined to neutralize the net charge in the nanoparticle core. The mixed solution was evaporated using a rotary evaporator under reduced pressure at 60°C to remove ethanol. PEG-PLE (2 mg) was then added to the flask and sonicated for 2 minutes to form self-assembled CFZ/tPNPs. CFZ/tPNPs were transferred into 1.5 mL conical tubes and freeze-dried. Our previous study showed that a 5% trehalose solution used as cryoprotectant performed best in maintaining the physiochemical properties of CFZ/tPNPs after freeze-drying compared to before⁷. Briefly, a 5% (w/v) trehalose solution was added to CFZ/tPNPs followed by flash freezing in dry ice (−78.5°C) for 4 h. Frozen CFZ/tPNPs was lyophilized using a Labconco FreeZone freeze drying system. The samples were lyophilized overnight at a primary drying temperature of −50°C and vacuum pressure of 0.02 mbar, followed by secondary drying phase of 25°C for 24 hours and maximum vacuum. Freeze-dried CFZ/tPNPs were weighed and stored in a −20°C freezer until use to extend the pharmaceutical stability of the formulation during storage.

Particle size measurement

The Zetasizer Nano ZS (Malvern, UK) was used to obtain particle characteristics. This instrument measures molecular size, molecular weight, particle size and zeta potential using dynamic light scattering (DLS). DLS was used for characterization of the nanoparticle in its liquid phase¹⁷. The freeze-dried tPNPs were reconstituted in PBS (10mM, pH 7.4) to a final concentration of 2 mg/mL to achieve this liquid phase. The suspension was exposed to an electromagnetic wave causing the light beam to scatter. From this scattering, a diffusion coefficient of the particles is determined with an equation f = kBT/6πηR_H to find the hydrostatic radius. The range of hydrostatic radius detected by DLS is between 0.3 nm to 10 μm, while parameters on the Zetasizer were set to 173° backscatter and 25°C temperature. The reconstituted sample was placed in a DTS 1070 cuvette for particle size analysis. Each sample was measured in triplicate and data was recorded as mean values and standard deviation.

Drug loading quantification

Freeze-dried CFZ/tPNP were reconstituted in saline. High performance liquid chromatography (HPLC) was used to determine the amount of CFZ in the nanoparticle. The mobile phase of the HPLC was an isocratic mixture of acetonitrile and water (ACN:water = 55:45, 1% formic acid, 0.5 mL/min, Agent Eclipse XDB-C18 column). Drug peaks were integrated at a wavelength of 210 nm and elution time was around 3.4 minutes. A calibration curve of known concentrations of CFZ (0.03 – 1 mg/ml) was used to determine the amount of CFZ loaded in the freeze-dried tPNPs. Encapsulation efficiency was calculated as the percentage of drug encapsulated to the drug added in preparation. Drug loading efficiency was defined as the percentage of drug by weight of drug encapsulated to the weight of tPNP. All samples were measured in triplicate and data were summarized by mean ± standard deviation.

Effects of buffers on particle stability and drug release kinetics

Freeze-dried CFZ/tPNPs were reconstituted in different buffers and the dissolved nanoparticles were first observed visually to see how fast they aggregated over time. Buffers used for stability assessment were tris buffered saline (TBS), phosphate buffered saline (PBS), normal saline (NS) and citrate buffer (CB). Particle size was also measured with DLS as described above to determine stability in these buffers. In parallel, drug release kinetics were analyzed for CFZ/tPNPs by using the dynamic dialysis method¹⁸. Briefly, freshly prepared tPNPs were reconstituted in potassium biphthalate sodium hydroxide buffer (pH 7.4, 5 mM) to a concentration of 10 mg/mL. Reconstituted CFZ/tPNPs were transferred to Thermo Scientific^™ Slide-A-Lyzer^™ MINI Dialysis Cups (20,000 MWCO) and placed in buffer (37°C, pH 7.4) to be dialyzed under sink conditions. At predetermined timepoints of 0, 0.5, 1, 3, 6, 24, 48, 72 h, samples were collected from the dialysis cups and drug remaining was determined by HPLC method described above. Drug remaining for each time point was normalized to the value obtained at t = 0 h. Drug release profiles were obtained by fitting the curve to an exponential decay using GraphPad Prism 9 software.

RESULTS

Preparation of CFZ/tPNPs using organic acids

Light scattering analysis of particle size showed tPNPs with less than 60 nm in diameter for all formulations with a size range between 33 nm and 55 nm after freeze drying and reconstitution. The average size of empty tPNPs was 46.1 ± 4.0 nm. CFZ precipitated after ethanol was evaporated without the inclusion of small molecule organic acids in our preparation. Therefore, organic acids were added to the formulations to prevent precipitation of CFZ from solution and enhance particle stability. Several organic acids were tested in preliminary studies, such as CA, LA, malic acid, maleic acid, succinic acid, and tartaric acid. Among these acids, CA, LA and tartaric acid were successful in preventing precipitation during ethanol evaporation. However, tartaric acid required extensive sonication after freeze-drying to facilitate complete dissolution of particles. tPNPs prepared using CA and LA prevented precipitation of CFZ and dissolved readily with vortexing. The incorporation of CA and LA did not adversely affect particle size distribution for all combinations. Average particle size after the addition of these organic acids and CFZ was 41.1 ± 8.7nm. The distribution was unimodal with a Gaussian profile. Figure 2 shows the particle size distribution for tPNPs with various combinations of CA and LA.

Figure 2.

Particle size distribution of nanoparticles

Effect of buffers on particle stability

CFZ/tPNPs were observed to see how fast they aggregated after reconstitution in various buffers. Tris buffer was unable to dissolve CFZ/tPNPs as indicated in its very large particle size (Figure 3). This is due to the amine groups in tris buffer disrupting the polyionic complex between HaβCD and PEG-PLE and breaking up the nanoparticles. PBS completely dissolved CFZ/tPNPs as no particles were seen in tube, but CFZ/tPNPs dissolved in PBS began to aggregate within 1 h after reconstitution and vortexing. Sonication was employed to enhance particle stability in PBS, but aggregation still occurred within 1 h. Citrate buffer and normal saline also completely dissolved CFZ/tPNPs. CFZ/tPNPs dissolved in citrate buffer and normal saline remained in solution for over 24 h.

Figure 3.

Particle stability in various buffers such as tris buffered saline (TBS), phosphate buffered saline (PBS), normal saline (NS) and citrate buffer (CB).

Drug loading quantification

HPLC analysis revealed that CFZ was successfully entrapped in organic acid stabilized tPNPs. The calibration curve from known concentrations of CFZ was used to determine drug concentrations. CFZ/tPNPs for all combinations of CA and LA showed a drug loading average above 1 mg/mL indicating a high encapsulation efficiency. Drug loading efficiency of all CFZ/tPNPs was about 13 wt%. Physiochemical properties of tPNPs are summarized in Table 1.

Table 1.

Physiochemical properties of CFZ/tPNPs

Formulation	CA(%):LA(%)	Particle size (nm)	PDI	Drug loading (mg/mL)
Empty tPNPs	-	46.1 ± 4.0	0.043	-
	100:0	33.7 ± 3.3	0.182	1.16 ± 0.23
	75:25	38.1 ± 5.8	0.313	0.94 ± 0.18
tPNPs with CFZ	50:50	55.8 ± 2.1	0.301	0.93 ± 0.05
	25:75	41.9 ± 7.9	0.364	1.06 ± 0.06
	0:100	36.1 ± 3.0	0.356	0.89 ± 0.16

Drug Release

All tPNPs prepared showed a biphasic drug release profile with an initial burst followed by sustained release shown as t_{1/2, fast} and t_{1/2, slow}, respectively (Figure 4). t_{1/2, fast} for initial burst release was evaluated within the first 1 h, followed by determining t_{1/2, slow} for slow drug release for 72 h. Figure 4 indicates that pure organic acids (100% CA or 100%LA) had the shortest burst release. The longest burst release was with 75% CA and 25% LA. Results with the inset displayed that using a ratio of organic acids produced greater efficacy in burst release reduction compared to pure organic acids. As summarized in Table 2, the half-life slow reduced as a function of CA proportion in tPNPs. Fast drug release was suppressed in all formulations except tPNPs with 100% LA, while half-life slow was delayed with the type or ratio of organic acids. tPNPs prepared with 100% CA had the longest half-life slow, and the formulation with 100% LA showed the shortest half-life slow.

Figure 4.

Drug release profiles for tPNPs with organic acids at various mixing ratios (enlarged view of inset for initial 6 h post incubation)

Table 2.

Kinetic parameters of CFZ release from tPNPs

Formulation	CA(%):LA(%)	Kfast(h−1)	Kslow(h−1)	t1/2, fast (h)	t1/2, slow (h)
tPNPs with CFZ	100:0	1.54	0.0239	0.45±0.11	29.06 ± 6.18
	75:25	4.09	0.0249	0.15±0.05	27.88 ± 4.34
	50:50	4.23	0.0275	0.18±0.10	26.29 ± 5.61
	25:75	4.46	0.0325	0.16±0.09	24.10 ± 3.81
	0:100	5.36	0.0402	0.13±0.07	17.26 ± 3.90

Effect of mixing ratio on drug remaining after 24h post incubation.

The percent of drug remaining at 24 h was greater for tPNPs with mixed CA:LA than tPNPs with single acid. tPNPs with higher CA% contained more CFZ. The plateau of the tPNP with 75%CA:25%LA contained the highest amount of CFZ after 24 h, with 50%CA:50%LA and the 25%CA/75%LA formulations following respectively (Figure 5). Nanoparticles containing single organic acids reduced the initial burst release but did not have as much drug remaining after 24 h. tPNPs containing 100% CA and 100% LA contained ~35% and ~57% respectively at 24 h. Although the burst release was lengthened, single organic acid tPNPs contained less than the ratioed organic acid tPNPs which contained between ~80–95% of drug after 1 h.

Figure 5.

Drug entrapment assessment (% of CFZ remaining at t = 24 h)

DISCUSSION

CFZ was approved by the FDA for the treatment of multiple myeloma due to the higher efficacy and lower toxicity than the first in class proteasome inhibitor Bortezomib¹⁹. Because of its success in multiple myeloma patients, CFZ has been further studied for the treatment of solid tumors and metastatic diseases. Despite promising preclinical data, CFZ achieved limited success in solid tumor treatment in the clinic potentially due to pharmaceutical limitations such as low solubility, poor biostability, and short release half-life, which lowered therapeutic outcomes^20–22. Previous studies suggest that nanoparticle formulations can improve therapeutic efficacy of small molecule drugs by fine-tuning the in vivo drug release rates^23–29, which would better control tumor accumulation, body clearance, and toxicity of various anticancer drugs^30–35. Unfortunately, few injection formulations are currently available that can safely increase drug retention in the body to maximize therapeutic potential and overcome clinical limitations of CFZ.

We previously reported that nanoparticle injection formulations made of tPNPs have great potential to address these problems because they can improve water solubility, biostability, and tumor accumulation of hydrophobic drugs including CFZ^{7, 36–40}. In this study, we confirmed that organic acid stabilized CFZ/tPNPs can prevent rapid degradation of the epoxyketone pharmacophore of CFZ and degradation of its tetrapeptide chain, which is beneficial to increase therapeutic efficacy of CFZ. We particularly assessed the potential utility of tPNPs to improve the entrapment and drug release half-life of CFZ.

tPNPs were designed by supramolecular interactions, comprising the formation of inclusion complex between CFZ and HaβCD followed by ionic interactions between amine groups of HaβCD and carboxylic groups of polyglutamate. These hydroxy acids enhance complexation of hydrophobic drugs with CDs compared to just binary complexation of drugs and CDs. This is because these acids modify the intramolecular hydrogen bond system involving the secondary hydrogen groups of CDs. Additionally, the hydroxy groups through hydrogen bonding allows the CD to have a more favorable interaction with surrounding water molecules thereby increasing solubility⁴¹. By utilizing organic acids such as CA and LA, we aimed to improve particle stability and extend drug release half-life of CFZ from tPNP. In some studies, CA has been shown to be effective in stabilizing nanoformulations^42–45. Three carboxylic acid groups of CA can stabilize the core of nanoparticles by surface adsorption and steric stabilization⁴³. LA has previously garnered some attention in biomedical research due to its involvement in cellular metabolism in the human body, and its ability to mitigate adverse reactions⁴⁶.

Our findings indicate that CFZ/tPNPs for all combinations of CA and LA maintained a ~50 nm particle size and inclusion of these additives did not adversely change particle size compared to drug-free tPNPs. Additionally, there was no significant difference in particle size across all combinations of CA and LA. This shows efficient entrapment of CFZ within the core of HaβCD and stabilization with the hydroxy groups of the organic acids. It must be noted that there was an increase in PDI for tPNPs where LA was included. The increase in PDI may be attributed to formation of PNPs becoming less stable as LA replaced CA, which have distinctive molecular structures and geometries in the nanoparticle core. Particle size is an important factor for nanoparticle biodistribution and circulation time in the body which determines therapeutic efficacy^47–48. Maintaining particle size below 50 nm is important because reports have shown that large nanoparticles (> 150 nm) are susceptible to plasma protein adsorption and opsonization leading to uptake by the liver^{35, 49}. On the other hand, sub 50 nm particles have been shown to have reduced blood clearance by macrophages^50–51.

Our results demonstrated a high drug loading of CFZ in tPNPs. All CFZ/tPNPs showed a drug loading approximately 1 mg/mL regardless of the CA to LA ratio. This translates to a very high encapsulation efficiency. Many nanoparticle systems have low to moderate encapsulation efficiency which limits the concentration of cytotoxic agents accumulating in tumors. CFZ/tPNPs have the potential to improve the accumulation of CFZ in solid tumors. The small size also allows it to have longer circulating time to offload its payload all together enhancing therapeutic efficacy.

We tested the stability of CFZ/tPNPs in different buffers. tPNPs were formed by the self-assembling process through ionic interactions between amine groups and carboxylic acid groups. CFZ/tPNPs dissolved in TBS showed poor stability as indicated by particle size. This is due to the competition between the amine group of TBS and the amine group on the surface of HaβCD, which disrupts the formation of ionic interaction needed for tPNPs. PBS performed better than TBS, but we observed aggregation of CFZ/tPNPs within 3 h of reconstitution in buffer. This is likely due to counter ions present in PBS slowly infiltrating and disrupting ionic interactions in tPNPs. NS and CB performed optimally in stabilizing tPNPs and remained stable over 24 h. Drug release profiles are important predictors of the stability and usefulness of drug delivery systems and how they modulate drug concentrations in vivo⁵². We aimed to improve the half-life of CFZ release from tPNPs. CA alone showed best performance in producing a sustained release of CFZ with a half-life of almost 30 h. This is a significant improvement over the current commercial CFZ formulation which has a half-life less than 1 h. Slow half-life reduced with increasing proportion of LA, and tPNPs made with 100% LA had the shortest slow half-life. However, CA and LA seemed to have a synergistic effect in reducing burst release. Using CA and LA at a 3 to1 ratio appeared to suppress the burst release 3-fold better than 100% CA. In addition, it has a comparable slow half-life (~ 28 h) to 100% CA. This 3 to 1 combination of CA to LA also showed the highest amount of drug remaining after 24 h, although the exact reason is unclear. One speculation is that four carboxylic acid groups in the combination might have improved ternary complexation in the nanoparticle core in comparison to the three on citric acid and the one on lactic acid. This indicates that the presence of LA reduces the amount of drug released in early time frame (< 24 h), leaving much of the drug available for sustained release. With 100% CA, a significant amount of the drug is lost due to burst release. With 100% LA burst release is mitigated, but the rapid release in the sustained phase reduces the amount of drug remaining after 24 h. Drug remaining after 24 h reduces as the proportion of CA in the formulation decreases. These results suggest that preparing CFZ/tPNPs with this particular combination of organic acids produced the most stable formulation.

CONCLUSION

tPNPs stabilized by CA and LA in mixture were found to improve the entrapment and drug release half-life for CFZ, which has few injection formulations for maximum therapeutic outcomes in vivo. The organic acid stabilized tPNPs formed sub-60 nm particles with > 1 mg/ml CFZ. The type and mixing ratio of organic acids appeared to mainly affect tPNP stability and drug release kinetics rather than charge density, which suggests that chemical structures of organic acids and drugs entrapped in the core of nanoparticle may play a major role in modulating entrapment and release of CFZ from tPNP. These findings are significant because organic acid-stabilized tPNPs having small particle size with high drug loading may expand the in vivo utility of CFZ and potentially other proteasome inhibitors from treating blood cancers to solid cancers.