Against the Flow: Delivering peptides to the blood through electrospray.

A team from the University of Portsmouth have recently published a study1 which may lay benchmarks for the production, dosage, and administration of electrosprayed nanoparticles which encapsulate peptide drugs. Given its accommodation of a wide range of test substances, and its reliable operation under a wide range of testing parameters, Spraybase was the team's tool of choice.

Peptides are strings of 3-30 amino amino acids. Those that act as hormones, are delivered throughout the body by the circulatory system. There would be countless therapeutic benefits to mimicking or supplementing a peptide's effects by delivering a sustained release of peptide after injection, but this has proven very difficult to achieve, even with electrohydrodynamic encapsulation techniques. Unlike the steroid class of hormones, peptides are easily damaged, and they tend to require encapsulation in hydrophilic substances, which do not interact with cell membranes. Nanofibers are not an option for blood vessels, and in vitro tissue cultures do not adequatley simulate the behaviour of nanoparticles in flowing blood.

Perhaps in light of these challenges, Ang-II (Human Angiotensin II) was used as a model peptide, not only because it has a direct and fast-acting physiological effect in vivo, raising blood pressure via blood vessel constriction, but because it is also known to stimulate a specific set of processes in cultured cell when tested in vitro. Since both effects of Ang-II are well-attested2, there would be firm grounds for the study to compare stationary and blood-borne formulations.

(NOSC structure adapted from3)

The team electrosprayed two encapsulation materials: NOSC (N-Octyl-O-Sulfate Chitosan) was used to form the encapsulation matrix, whilst tristearin (a triglyceride) was used in coaxial arrangements as the shell. NOSC is an artificial polymer, which comprises hydrophilic and lipophilic chemical groups bound to a natural polymer found in crustacean shells. It is therefore highly biocompatible4, and it can be combined with a variety of solvents and peptides, yet cause little chemical change in them. During the study, NOSC was either dissolved in DMSO (dimethyl sulfoxide) or in water, before being loaded with Ang-II. Using Spraybase, the team electrosprayed these Ang-II-loaded NOSC solutions with a single needle to form c.100 nm diameter 'matrix' particles. Through the coaxial needle, Ang-II-loaded NOSC was enveloped in tristearin which had been dissolved in DCM (dichloromethane). This saturated fat was used in the study because it is biocompatible, it preserves peptides, and it would enable the nanoparticle and its contents to access lipophilic cell membranes5.

Whereas NOSC dissolved in DMSO caused jet instabilities during coaxial electrospraying, its water-based counterpart successfully formed hollow nanoparticles of NOSC coated in tristearin, whose size, give or take 30nm, could be controlled by altering the distance between the needle and the collector. Where the collection distance was halved from 5cm to 2.5cm, the diameters of the coaxial nanoparticles increased from c.180 to c.270 nm..

Beside confirming the excellent (in this case c. 92%) encapsulation efficiency of electrospraying, Spraybase enabled the team to apply and control for a wide range of processing parameters. On noting that applying 30kV at at 5µL/min, despite producing small particles, caused significant degradation to Ang-II, the team could confidently reccommend an upper voltage limit of 20kV for Ang-II and peptides in general.

This study not only considered production methods, but examined how the coaxial nanoparticles interacted with living cells. When toxicity was assessed, it was determined that the particles, with or without Ang-II, did little to damage the cells' metabolic function in vitro except at very high doses. Even then, affected cells did not change structurally or functionally. The team also evaluated the coaxial particles’ release profiles in comparison to free Ang-II. These results raised many questions about how peptide-bearing nanoparticles respond to in vivo environments.

When the Ang-II-loaded nanoparticles were administered to a cell culture, they exhibited a triphasic release profile. In comparison to the burst of free Ang-II, the nanoparticle fromulation released only 5% of the Ang-II over the first five hours, possibly due to cracks in the tristearin shell. However, after those five hours, 15% of the Ang-II contained was burst-released. This burst was speculated to have been Ang-II held immediately beneath the tristearin layer or Ang-II that had been rapidly released and adsorbed by the NOSC solution during the atomisation and drying of the nanoparticles. The remaining 80% of the Ang-II was released over the following 15 hours. This release rate decayed in accordance with the decreasing surface area and Ang-II content of the NOSC nanoparticles.

The team also assessed the performance of these nanoparticles in a more realistic senario: in this case, the circulatory system of living trout. As before, the team compared nanoparticle-encapsulated and free Ang-II, this time through injection. As expected, the free Ang-II dose raised blood pressure sharply over 5 minutes before letting it fall over the following 25 minutes. Surprisingly, after the injection of Ang-II-loaded nanoparticles in the same fashion, the trout's blood pressure rose and peaked within 5 minutes, leaving a trace that was almost indistinguishable from the free Ang-II. However, following that, it was observed that the blood pressure declined slower than it had after the administration of free Ang-II, reflecting sustained Ang-II release. The difference between the release profile of free and nanoparticle-encapsulated Ang-II became significant by 25 minutes after administration. This was in marked contrast to the release profile of the nanoparticles in tissue culture. It was apparent that mechanical shearing stresses, and / or unknown chemical factors in the blood, had a substantial impact on the fate of the injected nanoparticles.

Besides establishing several rules of thumb which may guide the strategy of subsequent peptide electrospraying studies, these results strongly suggested that electrospraying has the potential to be a very valuable tool in the development of drug delivery systems. However, if that potential is to be realised, nanoparticle samples have to undergo relevant in vivo tests, in tandem with in vitro ones. This process would be facilitated by apparatus such as Spraybase, which, through its reliability, power, and flexibility, will enable coordinated research between groups, based on results that are highly comparable and reproducible, in order to better understand the fascinating interactions that can be engineered between living cells and electrosprayed nanomaterials.

  1. Rasekh M et al. J Mater Sci: Mater Med (2015) 26: 256. doi: 10.1007/s10856-015-5588-y
  2. Ruiz-Ortega M et al. Hypertension (2001) 38: 1382-1387 doi: 10.1161/hy1201.100589
  3. D.G.Fatouros et al. Nanoscale (2011) 3: 1218-1224 doi: 10.1039/c0nr00952k
  4. Zhang C et al Eur J Pharm Sci. (2008) 33(4-5): 415-23 doi: 10.1016/j.ejps.2008.01.012
  5. Jenning V, Lippacher A, & Gohla SH. J Microencapsul. (2002) 19(1): 1-10 doi:10.1080/713817583