Triggered release of therapeutic antibodies from nanodiamond complexes

Abstract

Recent reports have revealed that detonation nanodiamonds (NDs) can serve as efficient, biocompatible, and versatile drug delivery platforms. Consequently, further investigations exploring additional therapeutic applications are warranted. Current limitations associated with the non-specific nature of intravenous drugs limit the potential of certain pharmacological agents. One such treatment that could benefit from a stable delivery platform is antibody (Ab) therapy. Determination of Ab adsorption and desorption to a ND surface was subsequently examined using the transforming growth factor β (TGF-β) antibody as a model therapeutic. ND–Ab complexes were found to be stable in water through enzyme-linked immunosorbent assays (ELISAs), UV-vis spectroscopy and TEM, with no Ab released after ten days. Released Abs were detected in extreme pH solutions (3.5), DMEM (+) serum with pH levels ranging from 4 to 10.5, and inorganic saline solutions. Preserved activity of Abs released in DMEM (+) serum was confirmed using an ELISA. These results suggest ND–Ab complexes are synthesized and stabilized in water and are triggered to release active Abs upon exposure to physiological conditions.

The development of nanomaterials towards translational applications may help significantly improve efficacy and safety in the treatment of a spectrum of diseases that have proven challenging to address through conventional means. Efforts to improve current drug delivery mechanisms center on the ability to deliver therapeutics in a site-specific and controlled-release manner, as these are examples of essential properties that can reduce complications and side effects of treatment.¹ Therefore, a broad array of nanomaterials, such as carbon nanotubes, copolymer membranes, and gold nanoparticles, has been investigated to assess the efficacy of these drug-carrying vehicles.^2-7 Expanding upon the materials that have been investigated will undoubtedly broaden the strategies available towards enhanced pharmacological treatment.

A promising drug delivery platform that has recently been utilized towards versatile therapeutic delivery is based upon detonation nanodiamonds (NDs). These carbon-based particles integrate a comprehensive set of properties that may serve as a foundation for their future use in translationally relevant therapeutic applications. Studies completed thus far reveal that NDs possess biocompatible properties, as cells maintain integrity and morphology upon exposure to and incubation with NDs.^8-10 Moreover, NDs have high surface area to volume ratios allowing for significant loading capacities,¹¹ as well as functionalized surfaces allowing for chemical conjugation and adsorption of a variety of small molecules.^12-19 Insulin, DNA, siRNA, and insoluble chemotherapeutic drugs including purvalanol A and 4-hydroxytamoxifen have been successfully carried and delivered via NDs.^17-19 Additionally, evidence regarding the use of NDs as a drug delivery platform show that the ND–therapeutic complexes can preserve functional efficacy in vitro and in vivo.^13,16,20 Specifically, cellular assays performed suggest the retention of drug function after ND adsorption and release.¹³ These favorable properties indicate ND–drug complexes show promise for future drug delivery systems. As a result, further investigation concerning the therapeutic impact of NDs was performed.

A treatment regimen of clinical significance that may benefit from ND mediated delivery is antibody (Ab) therapy. The utilization of Abs as therapeutics has increased steadily over the past decade. The FDA currently has approved a number of Ab therapies, such as those for cancer, transplant rejection, and rheumatoid arthritis.^21-24 Additional Ab treatments are also in development, including anti-transforming growth factor β (TGF-β) for use in scarring/scleroderma^24,25 and additional cancer²⁶ applications. Previously, Abs have been covalently linked to NDs to detect pathogenic bacteria;¹⁵ however, efforts to use NDs as a means of delivering antibodies in a therapeutic context have yet to be explored. Therefore, this research focused on designing ND–Ab complexes that allow for Ab adsorption and desorption with preserved activity. Utilizing such ND–Ab complexes as a delivery platform may provide a promising new strategy for Ab therapy.

ND–Ab complexes were synthesized by combining NDs and Abs under dilute saline conditions. Prior to synthesis, solutions of NDs (NanoCarbon Research Institute Ltd., Nagano, Japan) were sonicated for 30 min. Mouse anti-human recombinant TGF-β₁ IgG Abs (R&D Systems; Minneapolis, MN) were reconstituted in 1× PBS as per manufacturer recommendation. ND–Ab solutions were incubated at room temperature for 15 min to facilitate adsorption. Isolation of ND–Ab complexes was completed through centrifugation (17 970 RCF for 2 h).

Ab adsorption onto NDs was determined by measuring remaining free Ab following centrifugation using an enzyme-linked immunosorbent assay (ELISA) specific to mouse IgGs (Alpha Diagnostic International; San Antonio, TX). In lieu of kit standards (mouse IgGs in serum), new standards were developed consisting of an anti-TGF-β₁ Ab gradient. Adsorption results completed via ELISA were validated through UV spectroscopy by measuring wavelengths indicative of Ab absorbance (280 nm) using a Beckman Coulter DU 730 Spectrophotometer (Beckman Coulter, Inc., Brea, CA). Test conditions and parameters (per manufacturer protocol) were conducted in triplicate, the mean and standard deviation of which are presented in all figures. ND–Ab complexes were also imaged via transmission electron microscopy (TEM). Separation through centrifugation (17 970 RCF for 2 h) provided a ND–Ab pellet which was subsequently rinsed with water and dried under vacuum. Samples were characterized using an FEI Tecnai G2 TEM at 200 kV.

ELISAs showed Ab adsorption onto the NDs through quantification of free Ab following ND–Ab complex isolation (Fig. 1A). Similar trends were observed with UV-vis analysis (280 nm) indicative of Ab concentration (Fig. 1B). TEM imaging of ND–Ab complex formation showed significant clustering with the ND–Ab complexes (Fig. 2A and B). Increased hydrodynamic particle size was further confirmed using associated dynamic light scattering assays. Of the ND–Ab ratios examined, a 2 : 1 mass ratio was identified as optimal and therefore used as a synthesis ratio for the remaining experimental trials.

Fig. 1.

(A) ELISA adsorption results. Addition of NDs to Ab solutions under dilute saline conditions showed a decreased amount of free Ab following ND–Ab complex isolation. As the ratio of NDs to Ab increased, more complexes formed thus further reducing the Ab concentration remaining in free solution. (B) UV-vis Ab adsorption results. Absorbance values taken at 280 nm indicative of protein concentration reveal Ab adsorption to NDs. These results confirm ND–Ab complex formation as indicated through ELISA.

Fig. 2.

TEM micrographs of ND–Ab complex synthesis. (A) Bare NDs. (B) ND–Ab complexes synthesized under dilute saline conditions.

Particle size and zeta potential measurements were also conducted. ND–Ab complexes were freshly prepared by mixing 62.5 μg of NDs with 62.5, 31.3 and 20.8 μg of Abs in 1250 μL of de-ionized water, corresponding to weight ratios ranging from 1 : 1 to 3 : 1. Solutions were incubated for 15 min at room temperature before measurement. Particle size and zeta potential measurements were conducted using the Zetasizer Nano ZS (Malvern, Worcestershire, UK). The size measurements were performed at 25 °C at a 173° scattering angle. The mean hydrodynamic diameter was determined by cumulative analysis. The zeta potential determinations were based on electrophoretic mobility of the nanoparticles in the aqueous medium, which was performed using folded capillary cells in automatic mode.

Unmodified NDs form complexes with an average size of 50 nm and a slightly positive surface (about 15 mV).¹⁸ Agglomeration was found to occur in free antibody solutions with an average size of 220 nm and a nearly negative zeta potential (Fig. 3A and B). The average size of the complexes formed was approximately 200–300 nm (Fig. 3A). The zeta potential of the ND–Ab aggregates increased with respect to the ND : Ab weight ratio and revealed an inherent stability compared to Ab alone (Fig. 3B). Thus, ND–Ab complexes may serve as a more effective delivery platform, as Ab loading and complex stability will undoubtedly aid in the efficient release and delivery of Abs.

Fig. 3.

(A) Relative size of ND–Ab complexes expressed as a weight ratio of ND : Ab. (B) Depiction of ND to Ab aggregate zeta potential measurements revealing innate stability of formed ND–Ab complexes compared to Ab alone. Increasing the amount of NDs does not significantly alter complex stability, supporting a 2 : 1 ratio as optimal to adsorb Ab to the ND surface.

To quantify antibody elution, initial separation through centrifugation (17 970 RCF for 2 h) allowed for elimination of unbound Ab. Remaining solvent was removed through aspiration, after which ND–Ab complexes were resuspended in the specified elution solvent and incubated accordingly. Quantification of Ab elution was accomplished through an additional centrifugation step, using similar methods as Ab adsorption quantification. Eluted Ab was quantified through a mouse IgG ELISA kit (Alpha Diagnostic). Elution was measured in de-ionized water, DMEM with 10% FBS and 1% pen/strep, and DMEM without FBS but with 1% pen/strep. The effect of pH was investigated by modifying media and non-media samples with HCl or NaOH to obtain a desired pH. In addition, the salt concentration was considered by measuring elution in varying concentrations of NaCl and NaHCO3, as well as a solution containing 81.9 mM NaCl, 44.05 mM NaHCO₃ and 5.33 mM KCl (concentrations of three most abundant inorganic salts in DMEM).

ND–Ab complexes were found to be stable in de-ionized water, with no Ab desorption from complexes after 1 or 10 days seen on ELISA (Fig. 4A). Similarly, no significant Ab release was observed from complexes in water solutions of modified pH from 4 to 12. However, the desorption of Abs did occur at a pH value of approximately 3.5, imparting insight into the processes responsible for ND–Ab complex formation and subsequent Ab release. The pH values at which Abs desorb coincided roughly with a pK_a of carboxyl groups that have been shown to be present on ND surfaces (approximately 4). Alterations in ND surface charges mediated by surrounding pH conditions may result in the introduction of opposite or like charges between the NDs and surrounding biomolecules that may result in binding, or release, respectively. In addition, the introduction of competing binding species to the ND surface may also facilitate antibody desorption, as discussed below. These observations support the notion that ND–Ab complex formation may be principally driven by electrostatic interactions between the ND surfaces and Abs.^14,27 Given the complex nature of the ND surface that has been previously examined, further studies into the interactions of ND surfaces with complex biomolecules such as antibodies/proteins may further elucidate the mechanisms of pH and solvent-mediated biomolecular binding and release.^28,29

Fig. 4.

ELISA elution results. (A) Elution in water as a function of pH. Complexes did not elute Abs in water after incubation for 1 or 10 days. Similarly, Abs were not released in water with pH values between 4 and 12. At the extreme pH of 3.5, Abs were released—as noted by an increase in Ab concentration. (B) Elution in media as a function of pH. Unlike elution in water, complexes readily eluted Abs in DMEM (+) serum with pH adjusted between 3.5 and 10.5. (C) Elution in DMEM (+) serum, DMEM (−) serum and inorganic salt solution consisting of three most abundant inorganic salts in DMEM after 1 day incubation. (D) Elution in inorganic salt solutions after 1 day incubation, as a function of salt concentration. Increasing NaCl and NaHCO₃ salt concentration has a positive effect on Ab elution, confirming the effect of inorganic salts on desorption. (E) Evaluation of eluted Ab activity after 1 day incubation. Concentration measurements from the activity independent ELISA and an activity dependent ELISA were similar, indicating no loss of anti-TGF-β₁ activity occurred during adsorption or elution. A decrease in reported concentration by the activity dependent ELISA when compared to the activity independent ELISA would have suggested a loss in Ab activity.

In contrast, ELISA analysis showed that ND–Ab complexes readily release Abs in DMEM (+) serum with adjusted pH values between 3.5 and 10.5 (Fig. 4B). This indicated that ND–Ab complex stability was influenced by the presence of media components. Competitive binding of media constituents to the ND surface may have served as a mechanism to mediate Ab binding and release. Additionally, the presence of various salts and/or buffers in media may alter charge properties of the ND surfaces and/or Abs that were responsible for complex formation. The observed differences between Ab release profiles in water versus DMEM (+) may be advantageous for clinical applications. These results suggest that it is possible to synthesize and store stable ND–Ab complexes in water, followed by a triggered release of Abs for the administration of complexes under physiological conditions.

Further analysis regarding the effect of various components of DMEM (+) serum was conducted to investigate the difference in desorption patterns. Comparisons of DMEM (+) serum and DMEM (−) serum indicated that serum significantly contributes to Ab desorption (Fig. 4C). Furthermore, an inorganic salt solution composed of the most abundant inorganic salts in DMEM also stimulated antibody release. As such, it was evident that the presence of inorganic salts contributed to antibody release within DMEM (−) serum. The positive effect that inorganic salts imposed on Ab release was confirmed through elution assays with varying concentrations of NaCl and NaHCO₃, where elution was found to increase with salinity (Fig. 4D). Release mediated by inorganic salts further supports the notion that ND–Ab complexes may form due to electrostatic interactions. As has been proposed with other nanoparticle–macromolecule complexes,^30-33 ionic salt components potentially displace bound Abs on the ND surfaces, effectively influencing the electrostatic forces responsible for the initial ND–Ab binding.

The activity of eluted anti-TGF-β₁ Abs was determined by comparing measured Ab concentration from two ELISAs. The first ELISA (mouse IgG kit utilized in adsorption/elution studies) used anti-mouse Abs specific to the constant region as the capture mechanism and therefore provided an anti-TGF-β₁ Ab concentration measurement independent of activity toward TGF-β₁. The second ELISA used TGF-β₁ as the capture mechanism and therefore provided an apparent Ab concentration that was affected by anti-TGF-β₁ Ab activity toward TGF-β₁. When comparing the reported concentrations from the two ELISAs, a lower concentration from the activity dependent, TGF-β₁ capture-method ELISA would have indicated a loss of activity.

For the TGF-β₁ capture-method ELISA, a rat anti-TGF-β₁ Ab from a TGF-β₁ ELISA kit (E-Bioscience; San Diego, CA) was adsorbed to a plate using the provided coating buffer. The plate was then incubated at room temperature with 25 μg mL⁻¹ TGF-β₁ (R&D Systems) for 1 h to saturate all adherent anti-TGF-β₁ Abs. The ELISA was subsequently carried out with the following reagents: anti-mouse HRP-conjugated Abs and TMB substrate (R&D Diagnostics), as well as wash and diluent solutions provided (Alpha Diagnostic mouse IgG ELISA kit).

Our results regarding the analysis of Ab functionality following elution from the ND complexes indicated that Ab activity was preserved during the adsorption/desorption process. Concentrations measured by the two ELISAs discussed above were found to be similar, indicating preserved Ab functionality during the adsorption and desorption processes (Fig. 4E).

Because of the broad spectrum of medical conditions that are currently being managed by therapeutic antibodies, including cancer and wound healing, this study introduced the use of NDs as platforms for rapid functionalization with therapeutic Abs, triggered Ab release, and preserved activity. The results presented suggest that ND–Ab complexes are a promising method of delivering Abs because of their ability to preserve Ab stability and release Abs under physiological conditions. The NDs were shown to bind readily with the anti-transforming growth factor β (TGF-β) antibody, which was utilized as a model therapeutic. The resulting complexes were observed to be stable in water, but released Abs upon administration in DMEM (+) serum at a broad range of pH levels. ELISA assays confirmed preserved Ab functionality. Due to the integrative properties possessed by NDs that are favorable towards drug delivery, the observed release profiles, and preserved activity demonstrated in this work, ND–Ab complexes may serve as promising Ab delivery platforms for enhanced therapeutic delivery and other applications in nanomedicine.