A review of solute encapsulating nanoparticles used as delivery systems with emphasis on branched amphipathic peptide capsules

Abstract

Various strategies are being developed to improve delivery and increase the biological half-lives of pharmacological agents. To address these issues, drug delivery technologies rely on different nano-sized molecules including: lipid vesicles, viral capsids and nano-particles. Peptides are a constituent of many of these nanomaterials and overcome some limitations associated with lipid-based or viral delivery systems, such as tune-ability, stability, specificity, inflammation, and antigenicity. This review focuses on the evolution of bio-based drug delivery nanomaterials that self-assemble forming vesicles/capsules. While lipid vesicles are preeminent among the structures; peptide-based constructs are emerging, in particular peptide bilayer delimited capsules. The novel biomaterial-Branched Amphiphilic Peptide Capsules (BAPCs) display many desirable properties. These nano-spheres are comprised of two branched peptides-bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK, designed to mimic diacyl-phosphoglycerides in molecular architecture. They undergo supramolecular self-assembly and form solvent-filled, bilayer delineated capsules with sizes ranging from 20 nm to 2 μm depending on annealing temperatures and time. They are able to encapsulate different fluorescent dyes, therapeutic drugs, radionuclides and even small proteins. While sharing many properties with lipid vesicles, the BAPCs are much more robust. They have been analyzed for stability, size, cellular uptake and localization, intra-cellular retention and, bio-distribution both in culture and in vivo.

Keywords: BAPCs; Drug delivery; Nanoparticle; Peptide bilayer; Self-assembly.

Figure 1

Classification of Nanocarrier Systems for Drug Delivery

Figure 2

Illustration of a micelle.

Figure 3. Polymeric micelle

Self-assembly and polymerization of a block copolymer yields a shell cross-linked knedel polymer assemblies, where the cross-linked outer shell can be decorated with biologically relevant ligands [Reprinted from Adv Drug Deliv. Rev, 56(11), Tu and Tirrell, Bottom-up design of biomimetic assemblies, 1537–1563, (2004) with permission from Elsevier]

Figure 4. Convergent and divergent synthesis of dendrimers and dendrons

Reprinted (adapted) with permission from Rosen et al. (2009), Chem Rev., 109(11), 6275–6540. Copyright 2009 American Chemical Society.

Figure 5. Illustration of a Liposome

By courtesy of Encyclopaedia Britannica, Inc., copyright 2007; used with permission. [http://www.britannica.com/EBchecked/media/92244/Phospholipids-can-be-used-to-form-artificial-structures-called-liposomes].

Figure 6. Electron microscopy pictures of POPC:POPE (6:4) liposomes made using extrusion

Seabra, M.B. (2006) Studies of a Channel-Forming Peptide Inserted into Liposomes formed by POPC:POPS and POPC:POPE, (M.S. Thesis Kansas State University).

Figure 7. Polymersomes derived from asymmetric block copolymers

Reprinted (adapted) with permission from Meng et al. (2009) Biomacromolecules, 10(2), 197–209. Copyright 2009 American Chemical Society.

Figure 8. Peptide amphiphile structure

The basic structural components of peptide nanoparticle components: (1) a hydrophobic tail, (2) lower, rigid part, including 4 cysteine residues (3) upper, flexible part, including 3 glycines, a phosphoserine group and an integrin-binding motif, RGD. "Reprinted (adapted) with permission from Tsonchev et al. (2004).

Figure 9. Snapshots from molecular simulations of peptide amphiphiles

“(a) The spherical micelle, (b) the micelle with β-sheets on the outside forming the corona, (c) the β-sheets, and (d) the fiber aggregate”. [Reprinted (adapted) with permission from Velichko et al. (2008), J. Phys Chem B., 112(8), 2326–34. Copyright 2008 American Chemical Society.]

Figure 10. Shear strength of peptide adhesives measured in Mega Pascals (MPa) with different tri-residue flanking the h₉ sequence

E, K and X represent the tripeptides (Glu)₃, (Lys)₃ and (diaminopropionic acid)₃, respectively. Peptide solutions-- 4% (360 μL) were spread onto marked 8.0 cm by 2.0 cm areas on one side of two separate strips of wood. The coated strips sat for 15 minutes at RT, before being pressed together for 5 minutes at 130 °C and 1.4 MPa • cm⁻¹.using a Hot Press Model 3890 Auto M (Carver Inc., Wabash, IN). The glued strips were then conditioned for 3 days at 50% relative humidity and 23 °C before being cut into test sized pieces. Reproduced from Shen et al.

Figure 11. TEM images of K₃FLIVIK₃ that were dried from peptide solutions prepared at different pH values

The left panel is peptide suspended at pH 12.0 and imaged at 70,000 x magnification. The center panel is peptide imaged at 34,000 x. The right panel is peptide dissolved at pH 7.0 and imaged at 70,000 x.

Figure 12. Graphic representation of the branched and linear amphipathic sequences initially tested

The color coding is added to signify those residues that are cationic (yellow), non-polar (blue) and hydrophilic (violet) along with the branch point (pink).

Figure 13. Transmission electron microscopy (TEM) obtained from the synthesis of nanocapsules solution (1.5 mM)

The image shows the process of fusing the nanocapsules, resulting in larger and more elongated structures (scale bar: 200 nm). Reproduced from Gudlur et al. (2012).

Figure 14

Scanning transmission electron microscopy (STEM) peptides labeled with Me-Hg 24 hours after mixing. Capsules were prepared with the peptides at 0.1 mM and 30% of them marked with Me-Hg. The images were captured using the inverted dark-field mode. Reproduced from Sukthankar et al.

Figure 15. Atomistic model of the BAPC peptide bilayer

In the center, the peptides are shown in a transparent surface, with Lys colored in yellow and other residues in blue and water in ball-and-stick mode. The top panel illustrates extensive π-π stacking interactions among Phe residues. The bottom panel shows the average electron density profiles of water (black line), Lys (red) and Phe residues (green line) calculated from last 40 ns of the 100 ns simulation.

Figure 16

Assembly protocol for peptides nanocapsules.

Figure 17. BAPC fusion time course

Peptides(0.1 mM) bis (FLIVI) and bis (FLIVIGSII) containing 30% Me-Hg were hydrated and samples were removed at the indicated times and dried. The STEM images are displayed in inverted dark field. The scale bar at the bottom of the micrographs are 200 nm, 500 nm, 200 nm, 100 nm, 200 nm and 500 nm, for the intervals of 0, 5, 10, 30, 60, and 120 min, respectively. Reproduced from Sukthankar et al.

Figure 18. BAPC Fusion Study

Panel A. Salt washed eosin Y trapped BAPCs were mixed with water filled BAPCs at 25 ° C. Fluorescence scans were taken at 5 min intervals for 235 min. The inset shows spectrum of sample stored at 4° C for 6.5 h. The units shown are identical to those in A. Panel B. Measured maximum eosin fluorescence intensity as a function of time during the fusion reaction. The t = 0 represents quenched value of salt washed eosin encapsulate in the capsules (2.2 mM). The data was fitted to a second order exponential with the error bars representing the SEM with n = 3. Reproduced from Sukthankar et al.

Figure 19. Fluorescence Microscopy images of BAPCs delivering TAMRA labeled protein to HeLa Cells

A) HeLa cells uptake of BAPCs carrying Tcytc; B) Control with Tcytc with Pep-1; C) BAPCs uptake containing TRNase; D) Control TRNase A with Pep-1. Reproduced from Sukthankar et al.

Figure 20. Time function of the BAPCs integrity

Images obtained with confocal microscopy of BAPC uptake by HeLa cells after 2 weeks showing: A) Excitation of Rhodamine in Dark field image; B) DIC image; C) Merge images exhibiting the Rhodamine inside of the BAPCs. Reproduced from Sukthankar et al.

Figure 21. Retention of BAPCs encapsulated with ²²⁵Ac. Encapsulation and retention of ²²⁵Ac within BAPCs over 7 days

Reproduced from Sukthankar et al.

Figure 22. Biodistribution of free and BAPC-encapsulated, ²²⁵Ac and its daughter ²¹³Bi, in CD1 mice

A) 1 h time point; B) 24 h time point. Reproduced from Sukthankar et al.