# Coating of Nanolipid Structures by a Novel Simil-Microfluidic Technique: Experimental and Theoretical Approaches

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## Abstract

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## 1. Introduction

## 2. Materials and Methods

#### 2.1. Materials

#### 2.2. Methods

#### 2.2.1. Production of Uncoated and Polycation-Coated Nanolipid Structures

#### 2.2.2. Z-potential and Size of Nanolipid Structures

#### 2.2.3. Transmission Electron Microscopy

#### 2.2.4. Mucoadhesiveness by Mucin Binding Assay

#### 2.2.5. Turbidimetry

#### 2.2.6. Stability during Storage

## 3. Results

#### 3.1. Adsorption of Polycations on a Nanolipid Structure Surface: Combining Experimental and Theoretical Approaches

#### 3.1.1. Experimental Approach to Nanolipid Structure Coating with a Polycation: Z-potential Evolution

#### 3.1.2. Theoretical Approach to a Nanolipid Structure Coating with a Polycation: Concepts of Saturation

_{Sat}, i.e., the minimum concentration of a polymer required to cover the oppositely charged particles, and the depletion concentration, C

_{Dep}*, the ceiling for the depletion flocculation [12]. These values are largely dependent on both the particle concentration and the polyelectrolyte properties, such as the radius of gyration, molecular weight, and concentration.

_{Sat}, from the Z-potential evolution, using an empirical model proposed by [13] for liposomes covered by chitosan:

_{0}is the zeta potential at zero chitosan concentration (uncoated liposomes), and Z

_{Sat}is the zeta potential at the saturation concentration C

_{Sat}. In this work, the proposed model for chitosan coating of liposomes was extended to the coating of vegan liposomes by cationic guar gum. By fitting Equation (1) to the experimental data (R

^{2}≥ 0.95), it was possible to obtain the values of Z

_{Sat}and C

_{Sat}for both standard liposomes and vegan liposomes (Table 1). In particular, the saturation concentration was 0.00537% w/v of chitosan for liposomes and 0.0057% w/v of cationic guar gum for vegan liposomes.

_{Sat}(kg/m

^{2}), can be calculated using an approach similar to that used in [11] for coating emulsion droplets with a biopolymer. In particular, for emulsions, Γ

_{Sat}can be calculated by the following equation:

_{PC,total}, and the mass of PC needed to form a single vesicle, M

_{PC,liposome}, as follows:

_{PC}is the PC density (1015 kg/m

^{3}at T = 298 K), and Δr is the thickness of the liposomal membrane, which is supposed to be around 4 nm [13]. Therefore, the surface coating at saturation, Γ

_{Sat}, for vesicles can be written as the mass of the polycation adsorbed per unit of the surface:

_{Sat}and M

_{PC,total}, their ratio can be re-written in terms of concentrations. Thus, Equation (5) becomes:

^{−7}kg/m

^{2}for liposomes and 1.06 × 10

^{−7}kg/m

^{2}for vegan liposomes (Table 1).

_{Dep}) required to promote depletion flocculation, as suggested by [12], for colloidal dispersions covered by oppositely charged polyelectrolytes:

_{A}is Avogadro’s number; v is the effective molar volume of the polyelectrolyte in the solution (in m

^{3}), calculated as $v=4\pi {r}_{\mathrm{PE}}{}^{3}/3$, with r

_{PE}as the effective radius of the polyelectrolyte molecules in the solution (Table 2); X is defined in the following equation according to [12]:

_{PE}) divided by the average distance between the surfaces of the particles, as shown in the following equation:

_{Sat}< C < C

_{Dep}*, particularly for liposomes covered by chitosan 0.00537 < C [% w/v] < 0.04840 and for vegan liposomes covered by cationic guar gum 0.00570 < C [% w/v] < 0.2282.

_{Ads}, which must be less than the time of the particle–particle collisions, τ

_{Col}, i.e., τ

_{Ads}/τ

_{Col}< 1. The critical polycation concentration (C

_{Ads}) is obtained when the adsorption time τ

_{Ads}is just equal to the time between the particle–particle collisions, τ

_{Col}[12]:

_{Ads}for both liposomes and vegan liposomes are shown in Table 2. If C > C

_{Ads}, the adsorption time is faster than the collision time between the particles, and, therefore, little vesicle aggregation occurs. Therefore, it should be possible to make stable multilayer-coated vesicles without flocculation by using intermediate polycation concentrations, such as C

_{Ads}< C < C

_{Dep}*. Lastly, for stable covered liposomes, the chitosan concentration should be between 0.0057 and 0.0484% w/v, and for stable covered vegan liposomes, the Guar HC concentration should be between 0.0086 and 0.2282% w/v.

#### 3.1.3. Combination of Experimental and Theoretical Approaches to Nanolipid Structure Coating with a Polycation

_{Sat}, C

_{Ads}, and C

_{Dep}* with the Z-potential evolution in Figure 2 (where it is visible that a constant value of Z-potential was reached for liposomes after adding 0.00625% w/v of chitosan, and for vegan liposomes by adding 0.005% w/v of cationic guar gum), the range of useful polycation concentrations was restricted to 0.00625%–0.01% w/v of chitosan for liposomes and 0.0089%–0.01% w/v of Guar HC for vegan liposomes.

#### 3.2. Characterization of Coated Nanolipid Structures

## 4. Conclusions

## Author Contributions

## Funding

## Acknowledgments

## Conflicts of Interest

## References

- Bochicchio, S.; Dalmoro, A.; Barba, A.; d’Amore, M.; Lamberti, G. New Preparative Approaches for Micro and Nano Drug Delivery Carriers. Curr. Drug Deliv.
**2017**, 14, 203–215. [Google Scholar] [CrossRef] [PubMed] - Bochicchio, S.; Dalmoro, A.; Bertoncin, P.; Lamberti, G.; Moustafine, R.I.; Barba, A.A. Design and production of hybrid nanoparticles with polymeric-lipid shell–core structures: Conventional and next-generation approaches. RSC Adv.
**2018**, 8, 34614–34624. [Google Scholar] [CrossRef] - Volodkin, D.; Mohwald, H.; Voegel, J.C.; Ball, V. Coating of negatively charged liposomes by polylysine: Drug release study. J. Control. Release
**2007**, 117, 111–120. [Google Scholar] [CrossRef] [PubMed] - Volodkin, D.; Ball, V.; Schaaf, P.; Voegel, J.C.; Mohwald, H. Complexation of phosphocholine liposomes with polylysine. Stabilization by surface coverage versus aggregation. Biochim. Et Biophys. Acta (BBA) Biomembr.
**2007**, 1768, 280–290. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Sybachin, A.; Efimova, A.; Litmanovich, E.; Menger, F.; Yaroslavov, A. Complexation of polycations to anionic liposomes: Composition and structure of the interfacial complexes. Langmuir
**2007**, 23, 10034–10039. [Google Scholar] [CrossRef] [PubMed] - Yaroslavov, A.A.; Sybachin, A.V.; Kesselman, E.; Schmidt, J.; Talmon, Y.; Rizvi, S.A.; Menger, F.M. Liposome fusion rates depend upon the conformation of polycation catalysts. J. Am. Chem. Soc.
**2011**, 133, 2881–2883. [Google Scholar] [CrossRef] [PubMed] - Horner, I.J.; Kraut, N.D.; Hurst, J.J.; Rook, A.M.; Collado, C.M.; Atilla-Gokcumen, G.E.; Maziarz, E.P.; Liu, X.M.; Merchea, M.M.; Bright, F.V. Effects of polyhexamethylene biguanide and polyquaternium-1 on phospholipid bilayer structure and dynamics. J. Phys. Chem. B
**2015**, 119, 10531–10542. [Google Scholar] [CrossRef] [PubMed] - Refai, H.; Hassan, D.; Abdelmonem, R. Development and characterization of polymer-coated liposomes for vaginal delivery of sildenafil citrate. Drug Deliv.
**2017**, 24, 278–288. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Mengoni, T.; Adrian, M.; Pereira, S.; Santos-Carballal, B.; Kaiser, M.; Goycoolea, F. A Chitosan—Based Liposome Formulation Enhances the In Vitro Wound Healing Efficacy of Substance P Neuropeptide. Pharmaceutics
**2017**, 9, 56. [Google Scholar] [CrossRef] - Dalmoro, A.; Bochicchio, S.; Lamberti, G.; Bertoncin, P.; Janssens, B.; Barba, A.A. Micronutrients encapsulation in enhanced nanoliposomal carriers by a novel preparative technology. RSC Adv.
**2019**, 9, 19800–19812. [Google Scholar] [CrossRef] [Green Version] - Guzey, D.; McClements, D.J. Formation, stability and properties of multilayer emulsions for application in the food industry. Adv. Colloid Interface Sci.
**2006**, 128, 227–248. [Google Scholar] [CrossRef] [PubMed] - McClements, D.J. Theoretical analysis of factors affecting the formation and stability of multilayered colloidal dispersions. Langmuir
**2005**, 21, 9777–9785. [Google Scholar] [CrossRef] [PubMed] - Laye, C.; McClements, D.; Weiss, J. Formation of biopolymer-coated liposomes by electrostatic deposition of chitosan. J. Food Sci.
**2008**, 73, N7–N15. [Google Scholar] [CrossRef] [PubMed] - Sandeep, C.; Deb, T.K.; Shivakumar, H. Cationic Guar Gum Poly Electrolytecomplex-Microparticles. J. Young Pharm.
**2014**, 6, 11. [Google Scholar] [CrossRef] - Erazo-Majewicz, P.; Modi, J.J.; Xu, Z.F. Cationic, oxidized Polysaccharides in Conditioning Applications. U.S. Patent 7,589,051, 25 September 2009. [Google Scholar]
- Dalmoro, A.; Bochicchio, S.; Nasibullin, S.F.; Bertoncin, P.; Lamberti, G.; Barba, A.A.; Moustafine, R.I. Polymer-lipid hybrid nanoparticles as enhanced indomethacin delivery systems. Eur. J. Pharm. Sci.
**2018**, 121, 16–28. [Google Scholar] [CrossRef] [PubMed] - Bochicchio, S.; Dalmoro, A.; Recupido, F.; Lamberti, G.; Barba, A.A. Nanoliposomes Production by a Protocol Based on a Simil-Microfluidic Approach. In Advances in Bionanomaterials; Springer: Berlin, Germany, 2018; pp. 3–10. [Google Scholar]
- Barba, A.A.; Lamberti, G.; d’Amore, M.; Bochicchio, S.; Dalmoro, A. Process for Preparing Nanoliposomes Comprising Micronutrients and Food Products Comprising Said Nanoliposomes. WO 2019/049186 A1, 6 september 2017. Patent WO 2019/049186 A1, 6 September 2017. [Google Scholar]
- Briuglia, M.L.; Rotella, C.; McFarlane, A.; Lamprou, D.A. Influence of cholesterol on liposome stability and on in vitro drug release. Drug Deliv. Transl. Res.
**2015**, 5, 231–242. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Tan, C.; Zhang, Y.; Abbas, S.; Feng, B.; Zhang, X.; Xia, S.; Chang, D. Insights into chitosan multiple functional properties: The role of chitosan conformation in the behavior of liposomal membrane. Food Funct.
**2015**, 6, 3702–3711. [Google Scholar] [CrossRef] - Magarkar, A.; Dhawan, V.; Kallinteri, P.; Viitala, T.; Elmowafy, M.; Róg, T.; Bunker, A. Cholesterol level affects surface charge of lipid membranes in saline solution. Sci. Rep.
**2014**, 4, 5005. [Google Scholar] [CrossRef] [Green Version] - Mady, M.M.; Darwish, M.M. Effect of chitosan coating on the characteristics of DPPC liposomes. J. Adv. Res.
**2010**, 1, 187–191. [Google Scholar] [CrossRef] [Green Version] - Risica, D.; Dentini, M.; Crescenzi, V. Guar gum methyl ethers. Part I. Synthesis and macromolecular characterization. Polymer
**2005**, 46, 12247–12255. [Google Scholar] [CrossRef] - Efimova, A.; Sybachin, A.; Yaroslavov, A. Effect of anionic-lipid-molecule geometry on the structure and properties of liposome-polycation complexes. Polym. Sci. Ser. C
**2011**, 53, 89. [Google Scholar] [CrossRef] - Morris, G.A.; Castile, J.; Smith, A.; Adams, G.G.; Harding, S.E. Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach. Carbohydr. Polym.
**2009**, 76, 616–621. [Google Scholar] [CrossRef] [Green Version] - Schatz, C.; Viton, C.; Delair, T.; Pichot, C.; Domard, A. Typical physicochemical behaviors of chitosan in aqueous solution. Biomacromolecules
**2003**, 4, 641–648. [Google Scholar] [CrossRef] [PubMed] - Wang, C.; Li, X.; Li, P.; Niu, Y. Interactions between fluorinated cationic guar gum and surfactants in the dilute and semi-dilute solutions. Carbohydr. Polym.
**2014**, 99, 638–645. [Google Scholar] [CrossRef] [PubMed] - Bhattacharya, S.; Haldar, S. Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain–backbone linkage. Biochim. Et Biophys. Acta (BBA) Biomembr.
**2000**, 1467, 39–53. [Google Scholar] [CrossRef]

**Figure 1.**Simil-microfluidic method piping representation according to the Italian UNICHIM (Association for the Unification of the Chemical Industry Sector) standard. ABOVE: Section of nanoliposome preparation: (

**1**–

**2**–

**3**) lipids/ethanol feed line; (

**4**–

**5**–

**6**) water feed line; (D-1 and D-2) feed tanks; (G-1 and G-2) peristaltic pumps; (I-1) injector (production section); (

**7**) water/ethanol nanoliposome suspension; (D-3) recovering/homogenizing tank; (Z-1) sonication element for homogenization and reducing vesicle size. Section of nanoliposome covering: (

**8**–

**9**–

**10**) nanoliposome suspension; (

**11**–

**12**–

**13**) polycation solution feed line; (D-4 and D-5) feed tanks; (G-3 and G-4) peristaltic pumps; (I-2) injector (production section); (

**14**–

**15**) coated nanoliposome suspension; (D-6) recovering/homogenizing tank. BELOW: The detail of interdiffusion between the lipid solution and the hydration solution (lines 3, 6, 7, and symbol I-1 in the UNICHIM representation) and between the liposome suspension and polycation solution (lines 13, 10, 14, and symbol I-2 in the UNICHIM representation) is given.

**Figure 2.**Zeta potential evolution for unloaded liposomes (

**A**) and vegan liposomes (

**B**) by increasing the polymer concentration from 0% (uncoated) to 0.01% (chitosan for liposomes, A; cationic guar gum for vegan liposomes, B). Symbols are used for experimental data; the line represents the model fitting of the experimental data.

**Figure 4.**(

**A**) Size (left; numerical distributions) and (

**B**) Z-average (right; intensity distribution) distributions of liposomes by increasing the chitosan concentration from 0% (uncoated) to 0.01%.

**Figure 5.**(

**A**) Size (left; numerical distributions) and (

**B**) Z-average (right; intensity distribution) distributions of vegan liposomes by increasing the cationic guar gum concentration from 0% (uncoated) to 0.01%.

**Figure 6.**TEM photos of both uncoated liposomes (

**A**) and liposomes coated with 0.01% w/v of chitosan (

**B**). The photos refer to larger vesicles for a better visualization of the liposome’s structure.

**Figure 7.**TEM photos of both uncoated vegan liposomes (

**A**) and those coated with 0.01% w/v of Guar HC (

**B**). The photos refer to larger vesicles for a better visualization of the liposome’s structure.

**Figure 8.**Mucoadhesiveness (

**A**) and stability measured by turbidimetry (

**B**) of uncoated liposomes and vegan liposomes and coated ones with 0.01% w/v of chitosan and guar HC, respectively.

**Figure 9.**Numerical size (

**A**), Z-Average (

**B**), PDI (

**C**), and Mucoadhesiveness (

**D**) evolution from 0 to 4 months in storage conditions of 4 °C for liposomes (squares) and liposomes coated with 0.01% w/v of chitosan.

**Figure 10.**Numerical size (

**A**), Z-Average (

**B**), PDI (

**C**), and Mucoadhesiveness (

**D**) evolution from 0 to 4 months in storage conditions of 4 °C for vegan liposomes (squares) and liposomes coated with 0.01% w/v of Guar HC.

**Table 1.**The values of Z

_{Sat}and C

_{Sat}, from the fitting between the experimental data of Z-potential and Equation (1), with a relevant coefficient of determination and Γ

_{Sat}.

Z_{Sat}, mV | C_{Sat}, % w/v | R^{2} | Γ_{Sat}, kg/m^{2} | |
---|---|---|---|---|

Liposomes | −19.1 | 0.00537 | 0.948 | 1.01 × 10^{−7} |

Vegan liposomes | −24.5 | 0.00570 | 0.990 | 1.06 × 10^{−7} |

**Table 2.**Values of C

_{Dep}* and C

_{Ads}for both standard liposomes and vegan liposomes, together with the values of molecular weight M and r

_{PE}of polycations.

M, kg/mol | r_{PE}, m | C_{Dep}*, % w/v | C_{Ads}, % w/v | |
---|---|---|---|---|

Liposomes–Chitosan | 310 | 4 × 10^{−9} [26] | 0.0484 | 0.0057 |

Vegan liposomes–Guar HC | 660 | 3 × 10^{−9} [27] | 0.2282 | 0.0086 |

**Table 3.**Numerical size, Z-average, and size distribution (PDI) of uncoated liposomes, vegan liposomes, and coated liposomes with 0.01% w/v of chitosan and guar HC, respectively.

Numerical Size (nm) ± SD | Z-Average (nm) ± SD | PDI ± SD | |
---|---|---|---|

Liposomes | 88.3 ± 19.0 | 252.8 ± 2.1 | 0.38 ± 0.02 |

Liposomes–Chitosan 0.01% | 257 ± 37.6 | 504.4 ± 12.1 | 0.34 ± 0.03 |

Vegan liposomes | 65.9 ± 14.2 | 179.5 ± 2.3 | 0.36 ± 0.00 |

Vegan liposomes–Guar HC 0.01% | 58.8 ± 1.3 | 174.5 ± 2.7 | 0.36 ± 0.01 |

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**MDPI and ACS Style**

Barba, A.A.; Bochicchio, S.; Bertoncin, P.; Lamberti, G.; Dalmoro, A.
Coating of Nanolipid Structures by a Novel Simil-Microfluidic Technique: Experimental and Theoretical Approaches. *Coatings* **2019**, *9*, 491.
https://doi.org/10.3390/coatings9080491

**AMA Style**

Barba AA, Bochicchio S, Bertoncin P, Lamberti G, Dalmoro A.
Coating of Nanolipid Structures by a Novel Simil-Microfluidic Technique: Experimental and Theoretical Approaches. *Coatings*. 2019; 9(8):491.
https://doi.org/10.3390/coatings9080491

**Chicago/Turabian Style**

Barba, Anna Angela, Sabrina Bochicchio, Paolo Bertoncin, Gaetano Lamberti, and Annalisa Dalmoro.
2019. "Coating of Nanolipid Structures by a Novel Simil-Microfluidic Technique: Experimental and Theoretical Approaches" *Coatings* 9, no. 8: 491.
https://doi.org/10.3390/coatings9080491