Charged Polypeptide Tail Boosts the Salt Resistance of Enzyme-Containing Complex Coacervate Micelles

[Image: see text] Encapsulation of proteins can have advantages for their protection, stability, and delivery purposes. One of the options to encapsulate proteins is to incorporate them in complex coacervate core micelles (C3Ms). This can easily be achieved by mixing aqueous solutions of the protein and an oppositely charged neutral-hydrophilic diblock copolymer. However, protein-containing C3Ms often suffer from salt-inducible disintegration due to the low charge density of proteins. The aim of this study is to improve the salt stability of protein-containing C3Ms by increasing the net charge of the protein by tagging it with a charged polypeptide. As a model protein, we used CotA laccase and generated variants with 10, 20, 30, and 40 glutamic acids attached at the C-terminus of CotA using genetic engineering. Micelles were obtained by mixing the five CotA variants with poly(N-methyl-2-vinyl-pyridinium)-block-poly(ethylene oxide) (PM2VP(128)-b-PEO(477)) at pH 10.8. Hydrodynamic radii of the micelles of approximately 31, 27, and 23 nm for native CotA, CotA-E20, and CotA-E40, respectively, were determined using dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). The encapsulation efficiency was not affected using enzymes with a polyglutamic acid tail but resulted in more micelles with a smaller number of enzyme molecules per micelle. Furthermore, it was shown that the addition of a polyglutamic acid tail to CotA indeed resulted in improved salt stability of enzyme-containing C3Ms. Interestingly, the polyglutamic acid CotA variants showed an enhanced enzyme activity. This study demonstrates that increasing the net charge of enzymes through genetic engineering is a promising strategy to improve the practical applicability of C3Ms as enzyme delivery systems

Enhanced stability of complex coacervate core micelles following different core-crosslinking strategies

Complex coacervate core micelles (C3Ms) are formed by mixing aqueous solutions of a charged (bio)macromolecule with an oppositely charged-neutral hydrophilic diblock copolymer. The stability of these structures is dependent on the ionic strength of the solution; above a critical ionic strength, the micelles will completely disintegrate. This instability at high ionic strengths is the main drawback for their application in, e.g., drug delivery systems or protein protection. In addition, the stability of C3Ms composed of weak polyelectrolytes is pH-dependent as well. The aim of this study is to assess the effectiveness of covalent crosslinking of the complex coacervate core to improve the stability of C3Ms. We studied the formation of C3Ms using a quaternized and amine-functionalized cationic-neutral diblock copolymer, poly(2-vinylpyridine)-block-poly(ethylene oxide) (QP2VP-b-PEO), and an anionic homopolymer, poly(acrylic acid) (PAA). Two different core-crosslinking strategies were employed that resulted in crosslinks between both types of polyelectrolyte chains in the core (i.e., between QP2VP and PAA) or in crosslinks between polyelectrolyte chains of the same type only (i.e., QP2VP). For these two strategies we used the crosslinkers 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and dimethyl-3,3′-dithiopropionimidate dihydrochloride (DTBP), respectively. EDC provides permanent crosslinks, while DTBP crosslinks can be broken by a reducing agent. Dynamic light scattering showed that both approaches significantly improved the stability of C3Ms against salt and pH changes. Furthermore, reduction of the disulphide bridges in the DTBP core-crosslinked micelles largely restored the original salt-stability profile. Therefore, this feature provides an excellent starting point for the application of C3Ms in controlled release formulations

Balancing Enzyme Encapsulation Efficiency and Stability in Complex Coacervate Core Micelles

Encapsulation of charged proteins into complex coacervate core micelles (C3Ms) can be accomplished by mixing them with oppositely charged diblock copolymers. However, these micelles tend to disintegrate at high ionic strength. Previous research showed that the addition of a homopolymer with the same charge sign as the protein improved the stability of protein-containing C3Ms. In this research, we used fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS) to study how the addition of the homopolymer affects the encapsulation efficiency and salt stability of the micelles. We studied the encapsulation of laccase spore coat protein A (CotA), a multicopper oxidase, using a strong cationic-neutral diblock copolymer, poly(N-methyl-2-vinyl-pyridinium iodide)-block-poly(ethylene oxide) (PM2VP128-b-PEO477), and a negatively charged homopolymer, poly(4-styrenesulfonate) (PSS215). DLS indeed showed an improved stability of this three-component C3M system against the addition of salt compared to a two-component system. Remarkably, FCS showed that the release of CotA from a three-component C3M system occurred at a lower salt concentration and over a narrower concentration range than the dissociation of C3Ms. In conclusion, although the addition of the homopolymer to the system leads to micelles with a higher salt stability, CotA is excluded from the C3Ms already at lower ionic strengths because the homopolymer acts as a competitor of the enzyme for encapsulation

Ekstraksi Dan Karakterisasi Serbuk Nano Pigmen Dari Daun Tanaman Jati (Tectona Grandis Linn. F)

Tanaman Jati (Tectona grandis linn. F) umumnya hanya dimanfaatkan bagian kayunya untuk industri meubel, namun bagian lain seperti daun kurang dimanfaatkan. Daun jati terutama bagian pucuk daun muda dapat menghasilkan pigmen. Produksi serbuk nano pigmen dari daun jati dan karakterisasi serbuk nano pigmen tersebut belum dilakukan. Tujuan penelitian ini adalah menghasilkan nano pigmen dari pucuk daun jati muda dalam bentuk serbuk dengan menggunakan persentase filler yang berbeda dan melakukan karakterisasi serbuk nano pigmen jati tersebut. Pucuk daun jati muda diberi perlakuan mekanik dengan penggerusan kemudian disaring, larutan yang diperoleh diukur partikelnya dengan Particle Size Analyzer (PSA), dan dikeringkan dengan penambahan filler maltodekstrin 5% dan 10%. Serbuk yang diperoleh dihitung rendemen, ukuran partikel, dan kelarutan dalam air. Warna merah yang dihasilkan dari filtrat pucuk daun jati muda berasal dari zat warna antosianin yang terkandung dalam daun jati muda. Ekstrak dari pucuk daun jati muda memiliki ukuran dengan kisaran 87,8- 318,1 nm dengan ukuran rata-rata 109,2 nm. Hal ini menunjukkan bahwa ekstrak tersebut merupakan produk nano di alam. Penambahan filler dengan konsentrasi berbeda berpengaruh terhadap warna, rendemen, ukuran partikel serbuk, dan kelarutan pigmen serbuk dalam air.

Ekstraksi Dan Karakterisasi Serbuk Nano Pigmen Dari Daun Tanaman Jati (Tectona Grandis Linn. F)

Tanaman Jati (Tectona grandis linn. F) umumnya hanya dimanfaatkan bagian kayunya untuk industri meubel, namun bagian lain seperti daun kurang dimanfaatkan. Daun jati terutama bagian pucuk daun muda dapat menghasilkan pigmen. Produksi serbuk nano pigmen dari daun jati dan karakterisasi serbuk nano pigmen tersebut belum dilakukan. Tujuan penelitian ini adalah menghasilkan nano pigmen dari pucuk daun jati muda dalam bentuk serbuk dengan menggunakan persentase filler yang berbeda dan melakukan karakterisasi serbuk nano pigmen jati tersebut. Pucuk daun jati muda diberi perlakuan mekanik dengan penggerusan kemudian disaring, larutan yang diperoleh diukur partikelnya dengan Particle Size Analyzer (PSA), dan dikeringkan dengan penambahan filler maltodekstrin 5% dan 10%. Serbuk yang diperoleh dihitung rendemen, ukuran partikel, dan kelarutan dalam air. Warna merah yang dihasilkan dari filtrat pucuk daun jati muda berasal dari zat warna antosianin yang terkandung dalam daun jati muda. Ekstrak dari pucuk daun jati muda memiliki ukuran dengan kisaran 87,8- 318,1 nm dengan ukuran rata-rata 109,2 nm. Hal ini menunjukkan bahwa ekstrak tersebut merupakan produk nano di alam. Penambahan filler dengan konsentrasi berbeda berpengaruh terhadap warna, rendemen, ukuran partikel serbuk, dan kelarutan pigmen serbuk dalam air

Effect of enzymatic cross-linking of naringenin-loaded β-casein micelles on their release properties and fate in in vitro digestion

The microbial transglutaminase (mTG) was used to improve the stability of the naringenin-loaded β-casein micelles (CNMs). The formation of cross-linked CNMs was confirmed by SDS-PAGE electrophoresis, showing a decrease in monomeric β-CN levels with increasing crosslinking time. Dynamic light scattering (DLS) showed that after crosslinking the particle size distribution did not change upon dilution, suggesting occurrence of intra-crosslinking. Fluorescence spectroscopy and circular dichroism (CD) showed that crosslinking induced only minor changes in the structure. Finally, release of naringenin in buffer at pH 7.4 demonstrated a slower release from the cross-linked micelles compared to the untreated micelles. In addition, the cross-linked micelles exhibited a partial resistance to pepsin enzyme. We conclude that crosslinking with mTG is a suitable method to modulate naringenin release kinetics from β-CN micelles and improves the potential of these micelles as delivery systems targeted to the small intestine.</p

Ekstraksi Dan Karakterisasi Serbuk Nano Pigmen Dari Daun Tanaman Jati (Tectona Grandis Linn. F)

Tanaman Jati (Tectona grandis linn. F) umumnya hanya dimanfaatkan bagian kayunya untuk industri meubel, namun bagian lain seperti daun kurang dimanfaatkan. Daun jati terutama bagian pucuk daun muda dapat menghasilkan pigmen. Produksi serbuk nano pigmen dari daun jati dan karakterisasi serbuk nano pigmen tersebut belum dilakukan. Tujuan penelitian ini adalah menghasilkan nano pigmen dari pucuk daun jati muda dalam bentuk serbuk dengan menggunakan persentase filler yang berbeda dan melakukan karakterisasi serbuk nano pigmen jati tersebut. Pucuk daun jati muda diberi perlakuan mekanik dengan penggerusan kemudian disaring, larutan yang diperoleh diukur partikelnya dengan Particle Size Analyzer (PSA), dan dikeringkan dengan penambahan filler maltodekstrin 5% dan 10%. Serbuk yang diperoleh dihitung rendemen, ukuran partikel, dan kelarutan dalam air. Warna merah yang dihasilkan dari filtrat pucuk daun jati muda berasal dari zat warna antosianin yang terkandung dalam daun jati muda. Ekstrak dari pucuk daun jati muda memiliki ukuran dengan kisaran 87,8- 318,1 nm dengan ukuran rata-rata 109,2 nm. Hal ini menunjukkan bahwa ekstrak tersebut merupakan produk nano di alam. Penambahan filler dengan konsentrasi berbeda berpengaruh terhadap warna, rendemen, ukuran partikel serbuk, dan kelarutan pigmen serbuk dalam air. </div