30th Annual Conference of the European Society for Biomaterials together with the 26th Annual Conference of the German Society for Biomaterials (DGBM)
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Drug delivery 3

Session chair: Uziel , Almog , (Shenkar – Engineering Art Design, Department of Polymers and Plastics Engineering, Ramat-Gan, Israel); von Recum , Horst A. , (Case Western Reserve University, Cleveland, US)
 
Shortcut: IX-OS35
Date: Thursday, 12 September, 2019, 4:45 p.m.
Room: Conference room 2+3
Session type: Oral

Contents

Click on an contribution to preview the abstract content.

4:45 p.m. IX-OS35-01

Curcumin attenuates effects of (reduced)-Graphene Oxide tetrapodal networks as novel implant materials on cytotoxicity, inflammation & glial scarring in murine acute brain slices and human cell lines in vitro (#237)

C. Schmitt1, F. Rasch2, F. Schütt2, Y. Kumar2, M. Lohe3, A. S. Nia3, X. Feng3, R. Lucius1, J. Held-Feindt4, R. Adelung2, K. Hattermann1

1 University of Kiel, Institute of Anatomy, Kiel, Schleswig-Holstein, Germany
2 University of Kiel, Institute for Materials Science, Kiel, Germany
3 TU Dresden, Chair for Molecular Functional Materials and Center for Advancing Electronics Dresden , Dresden, Germany
4 UKSH Kiel, Department of Neurosurgery, Kiel, Germany

On behalf of GRK-2154 Materials for Brain

Introduction

For neurological diseases such as glioblastoma multiforme or epilepsy, brain implants are promising tools for the direct drug application to overcome systemic toxicity. However, the possibility to surface-functionalize the scaffold material as well as the electrical conductivity are often important requirements for neural implants, offering the possibility of either a targeted or a triggered, on-demand drug delivery [1]. Respecting this, Graphene Oxide (GO), a derivate of Graphene (G) with a high surface area has gained attention, since it consists of a single atomic carbon layer, decorated with hydrophilic functional groups [3] that can interact with peptides and proteins via chemical or physical bonding [4], therefore making it easy to bio-functionalize. However, GO is an insulator so that in cases where the conductivity of the material is more important than the functionalization, the oxygen content of GO can be reduced by several techniques to produce the highly conductive material reduced GO (rGO) [5,6]. Therefore GO and rGO are two materials of interest for neuro implant development.

Though, the surgery as well as the implant material can elicit a harmful tissue response causing an alteration of the neuro-chemical environment of the brain and therefore leading to the formation of a glial scar. Beside its adverse effects on neural circuits and plasticity, this scar tissue has influence on the efficacy of the implant, since it can reduce and alter the release of the drug from the implant as well as its conductivity. Thus, neural tissue reactions towards GO and rGO implant materials need to be investigated in suitable in vitro models [7,8].

Here we show the indirect response of different in vitro models towards highly porous tetrapodal rGO and GO networks [publication/manuscript in progress, Rasch et al.]. The anti-inflammatory and –fibrotic compound Curcumin was used in co-stimulation with the implant materials, in order to inhibit possible inflammation and glial scarring reaction towards the materials.

Experimental Methods

The macroscopic GO and rGO networks have been produced by wet chemical infiltration of highly porous (94% porosity) ceramic templates [9] with a GO dispersion. Afterwards, the template was removed by chemical etching in 1 M HCl for 12 hours followed by washing in ethanol and critical point drying of the networks. For fabrication of rGO scaffolds, the GO networks were reduced in ascorbic acid prior to template removal. GO and rGO materials were then studied for their cytotoxicity (Cytotox Fluor and WST-1 assay) as well as inflammatory and glial scarring responses to different brain in vitro models in co-stimulation with curcumin. Therefore, human astrocytes or microglia (SVGA respective HMC3 cell lines) as well as murine acute brain slices were stimulated indirectly (materials were placed in 0.4µm cell culture inserts) with the materials or material-eluate media, regarding ISO-10993-5. In respect of the acute and chronic reaction towards the implant, cells or brain slices were incubated for up to 6 days. Their material-specific reactions were studied on RNA or protein level.

Results and Discussion

Human astrocyte and microglia cell lines did not show any alterations in proliferation or cell death when incubated indirectly with GO and rGO materials for 24h. However, murine acute brain slices showed slightly elevated cytotoxicity after 6 days of indirect incubation with materials. Cytotoxic effects were less pronounced upon incubation with rGO in comparison to GO, and further alleviated by curcumin. Glial scarring associated markers such as glial fibrillary acidic protein (GFAP), tenascin, fibronectin, nestin or vimentin as well as inflammatory markers (e.g. interleukin-6) were induced in murine acute brain slices upon 6 days incubation with materials. Interestingly, these effects were clearly higher upon incubation with rGO than with GO, and could again be eased by curcumin co-stimulation. These results could be sustained by corresponding findings in human astrocyte and microglia cultures.

Conclusion

The results indicate that GO and rGO have a rather low impact on cytotoxicity or proliferation of human neural cell lines and murine acute brain slices, when cultivated indirectly with the materials for different time periods. However, reducing the oxygen amount of GO to obtain rGO seems to influence significantly the glial scarring and inflammatory responses in vitro, since the effects on cells and brain slices are more dominant after incubation with rGO. All investigated effects could be diminished by co-stimulation with curcumin, an anti-inflammatory and anti-fibrotic drug. Using these different in vitro models and by co-stimulating with curcumin we are currently trying to understand these effects more deeply and by this, hoping to improve the implant material design to reduce tissue reactions towards them.

References

[1] Servant, A.; Leon, V.; Jasim, D.; Methven, L.; Limousin, P.; Vazquez Fernandez‐Pacheco, E.; Prato, M., Kostarelos, K. Graphene‐Based Electroresponsive Scaffolds as Polymeric Implants for On‐Demand Drug Delivery. Advanced Healthcare Materials, 2014, p 1334-1343

 [2] Aliofkhazraei, M.; Ali, N.; Milne, W. I.; Ozkan, C. S.; Mitura, S.; Gervasoni, J. L. Graphene Science Handbook: Electrical and Optical Properties; CRC Press, 2016; p 715.

[3] Shin, S. R.; Li, Y. C.; Jang, H. L.; Khoshakhlagh, P.; Akbari, M.; Nasajpour, A.; Zhang, Y. S.; Tamayol, A.; Khademhosseini, A. Graphene-Based Materials for Tissue Engineering. Adv. Drug Delivery Rev. 2016, 105, 255– 274, DOI: 10.1016/j.addr.2016.03.007

[4] Smith, S. C.; Ahmed, F.; Gutierrez, K. M.; Frigi Rodrigues, D. A Comparative Study of Lysozyme Adsorption with Graphene, Graphene Oxide, and Single-Walled Carbon Nanotubes: Potential Environmental Applications. Chem. Eng. J. 2014, 240, 147– 154, DOI: 10.1016/j.cej.2013.11.030

[5] Dreyer, D.R.; Park, S.; Bielawski, C.W.; Ruoff, R.S., Chem. Soc. Rev., 39 (2010), pp. 228-240

[6] Dave, S.H.; Gong, C.; Robertson, A.W.; Warner, J.H.; Grossman, J.C., ACS Nano, 10 (2016), pp. 7515-7522

[7] Kristensen, B.W.; Biocompatibility of silicon-based arrays of electrodes coupled to organotypic hippocampal brain slice cultures. Brain Research, 896, 1-17, 2001

[8] Schommer, J.; et al; Method for organotypic tissue culture in the aged animal. MethodsX, 4, 166-171, 2017

[9] Mishra, Y.; Kaps, S.; Schuchardt, A., Paulowicz, I.; Jin, X.; Gedamu, D.; Freitag, S.; Claus, M., Wille, S.; Kovalev, A.; Gorb, S.N.; Adelung, R.; Fabrication of Macroscopically Flexible and Highly Porous 3D Semiconductor Networks from Interpenetrating Nanostructures by a Simple Flame Transport Approach; Particle & Particle Systems Characterization 2013, 30, 9, 775-783

Keywords: A-06 a - Biomaterials for drug delivery, A-08 a - Biocompatibility, A-09 b - Immunomodulatory biomaterials
5:00 p.m. IX-OS35-02

Collagen-based multilayer films for wound healing (#53)

M. H. Iqbal1, F. Boulmedais1, F. Meyer2

1 Centre National de la Recherche Scientifique, CNRS UPR 22, Institut Charles Sadron, Strasbourg Cedex 2, France
2 Université de Strasbourg, Institut National de la Santé et de la Recherche Scientifique, UMR_S 1121Biomaterials and Bioengineering, Strasbourg Cedex, France

Introduction

Development of multifunctional materials is mandatory to challenge complex physiological processes like wound healing. Indeed, wound healing, especially in open wounds, can face infection or pathophysiological condition, like diabetes, that can impede the healing process. Collagen is the main component of the extracellular matrix comprising up to 30 % of human skin. Several studies have shown that collagen can induce wound healing by promoting the recolonization and the proliferation of epithelial cells with low antigenicity, and low inflammatory properties [1, 2]. However, tissue’s matrix metalloproteases (MMPs) and bacterial infection may result in the enhancement of the breakdown of collagen-based material [3, 4]. Tannic acid (TA) is a polyanionic polyphenol extracted from plants possessing antimicrobial, antioxidant and anti-inflammatory properties. It can form complexes with proteins through H-bonding that can drive the build-up process and further becoming a protective layer of the epithelial tissue [5, 6]. Though TA has shown ample popularity for material processing (crosslinking) and antimicrobial activity, its cytotoxicity towards eukaryotic cells should not be neglected [7].

Herein, we develop collagen/TA films using the layer-by-layer method (fig 1a) in order to support wound healing in harsh conditions.

Experimental Methods

Collagen/TA films were developed by dip assisted layer-by-layer assembly (fig 1a) at acidic pH. The buildup was followed by Quartz Crystal Microbalance with Dissipation (QCM-D). These films were further characterized by Atomic Force Microscopy (AFM), Scanning Electron Microscopy (FESEM), Fourier Transform Infrared Spectroscopy (ATR), Circular Dichroism (CD) Spectroscopy, and Isothermal Titration Calorimetry (ITC). Their biological characterization includes stability in physiological buffers at pH 7.4, antimicrobial activity against Staphylococcus Aureus and cytotoxicity towards various fibroblast cell lines.

Results and Discussion

Collagen/TA films have an exponential growth observed by QCM-D and a fibrillar topography observed by AFM (Fig 1b). FTIR and CD experiments showed that collagen keeps its native structure. Such film, build in acidic pH, is stable up to 72 h in a buffer solution at pH 7.4 and releases TA in solution until 25 µg/mL in PBS. The films show release-killing property towards Staphylococcus Aureus without cytotoxicity for potential host cell like fibroblasts. Noteworthy, different buffers were tested for the construction of the film. Buffer type has emerged as a pivotal parameter leading to dissimilar physico-chemical and biological properties. ITC measurements showed that it could be related to the modification of the interaction between collagen and tannic acid in different buffers.

Conclusion

This work shows that the physico-chemical optimization of tannic acid based films is of paramount importance for future use of tannic acid in material production and their biomedical applications.

References

[1]        D. Brett, "A Review of Collagen and Collagen-based Wound Dressings," Wounds, vol. 20, 2008.

[2]        V. Natarajan et al., "Preparation and properties of tannic acid cross-linked collagen scaffold and its application in wound healing," Journal of Biomedical Materials Research Part B, vol. 101, 2013.

[3]        A. Yahyouche et al., "Macrophage-mediated degradation of crosslinked collagen scaffolds," Acta Biomaterialia, vol. 7, 2011.

[4]        S. Chattopadhyay et al., "Collagen-Based Biomaterials for Wound Healing," Biopolymers, vol. 101, 2014.

[5]        P. Velmurugan et al., "Investigation on interaction of tannic acid with type I collagen and its effect on thermal, enzymatic, and conformational stability for tissue engineering applications," Biopolymers, vol. 101, 2014.

[6]        A. de Sousa Leal et al., "Incorporation of tannic acid in formulations for topical use in wound healing: A technological prospecting," African Journal of Pharmacy and Pharmacology, vol. 9, 2015.

[7]        F. Reitzer et al., "Polyphenols at interfaces," Advances in Colloid and Interface Science, vol. 257, 2018.

[8]        G. Decher, "Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites," Science, vol. 277, 1997.

Acknowledgement

M.H.I acknowledges the Higher Education Commission’s (Pakistan) Ph.D. Fellowship program “HRDI-UESTPs/UETs Phase 1, Batch-V” for sponsoring the Doctorate studies. The authors acknowledge Institut Carnot MICA for financial support in the framework of DIAART project.

Figure 1:
A) Schematic representation of Layer-by-Layer technique based on the alternated deposition of oppositely charged polyelectrolytes obtained by the dipping process [8] (B) shows the topography of Collagen/tannic acid film observed by Atomic Force Microscopy (AFM).
Keywords: A-02 b - Coatings, A-07 b - Antibacterial, A-08 a - Biocompatibility
5:15 p.m. IX-OS35-03

Physical immobilization of particles inspired by pollination (#438)

L. F. Santos1, A. S. Silva1, C. R. Correia1, J. F. Mano1

1 University of Aveiro, Aveiro Institute of Materials, Aveiro, Portugal

Introduction

Transdermal drug delivery patches for cutaneous wounds treatment induce a faster healing of the wound by delivering therapeutic agents that are included over or inside the patch. Commercially available patches can carry limited amounts of drugs, which are already incorporated within the patch, not allowing for a personalized formulation. Thus, it is crucial the development of improved drug patches with higher therapeutic dose. The unique features exhibited by biological organisms in nature have been a source of inspiration for the development of high-performance structures and biomaterials for the delivery of therapeutic agents.[1] Honey bees for instance, present a peculiar hairy structure that allows them to fix and carry millions of pollen particles. The pollen grains, which are of a similar diameter to the spacing between the hairs, are entrapped and temporarily retained, only being released on movement of the creature’s leg.[2] Replicating this natural structure using microfabrication could provide a substrate to be used in a wide variety of applications that require simple, non-permanent, high-content and purely physical immobilization of solid particulate objects. Drug delivery systems are one potential application of these biomimetic substrates. Inspired by this, we proposed the concept of a micropatterned surface featuring high aspect ratio elastic micropillars spaced to mimic the hairy surface of bees. We explored the applicability of such surface as patch, to be used in wound healing, able efficiently entrap high amounts of drug particles at the microscale and release them in a controlled and sustained manner.

Experimental Methods

The hypothesis was validated by investigating the ability of polydimethylsiloxane (PDMS) microfabricated patches to fix microparticles. The patches were fabricated by soft lithography [4] and characterized by SEM, fluorescent microscopy and tensile strength. The geometrical arrangement, spacing, height and flexibility of the fabricated micropillars, and the diameter of the microparticles, were investigated.

Our biomimetic surfaces were explored for their ability to fix solid microparticles for drug-release applications, using tetracycline hydrochloride as a model antibiotic. The release profile and antimicrobial activity were herein determined using patches with both tetracycline powder and tetracycline alginate microparticles.

Results and Discussion

Inspired by the ability of the hairy structure of bees to entrap millions of pollen grains, we developed a flexible polymer substrate that replicated this extremely efficient system.[5] Higher entrapment capability was found through the match between particle size and pillar spacing, being consistent with the observations that the diameter of pollen grains is similar to the spacing between hairs on bees’ legs. Moreover, taller pillars (analogous to the high aspect ratio of the bees’ hairs) permitted immobilization of higher quantities of particles. These novel surfaces allowed fixation of more than 20 mg/cm2 of antibiotic and interestedly, this value is similar to the capture of pollen grains by bees – 27 mg. Moreover, such surfaces presented a dose significantly higher - about 5 times more - than currently available patches (5.1 mg/cm2) approved by FDA,[6] providing high drug concentration that could solve the current problems associated with passive drug delivery patches. We also verified that both solid drug powder and hydrogel microparticles could be immobilized in the proposed substrates, indicating the great potential of these systems to be used as patches for drug delivery.

Conclusion

The results herein described suggest that the proposed textured surfaces could be further considered for the development of high-performance patches for clinical applications, through simple contact of the patch with the powdered product. We hypothesize that such devices could be easily explored to wide variety of applications that required large quantities of microparticles in biomedical and biotechnological fields.

References

1.          Sanchez, C.et al. Nat. Mater. 4, 277–288 (2005).

2.          Amador, G. J. et al. 12, 1–11 (2017).

3.          Wiedersberg, S. et al. J. Control. Release 190, 150–156 (2014).

4.          Gitlin, L. et al. Lab Chip 9, 3000–3002 (2009).

5.          Santos, L. F. et al. Proc. Natl. Acad. Sci. 201813336 (2019).

6.          Santos, L. F. et al. Eur. J. Pharm. Sci. 118, 49–66 (2018).

Acknowledgement

The authors acknowledge the financial support by the European Research Council grant agreement ERC-2014-ADG-669858 for project “ATLAS”. The work was developed within the scope of the project CICECO-Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT Ref. UID /CTM /50011/2013), financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement.

Schematic illustration of the proposed biomimetic micropatterned patch

A) The bees’ body is covered with hairs. Pollen grains are caught between the bees’ hair, and their size is similar to the hair spacing. Image courtesy of Charles Krebs (photographer).

(B) A bioinspired patch can be developed through the construction of a micropatterning structure with micropillars with controlled spacing (s) and height (h).

(C) Capture of solid microparticles in a flexible substrate featuring well-organized micropillars by direct contact and pressure; we hypothesized that the entrapment effectiveness will depend on the relationship between the pillar spacing (s) and the size of the microparticles.

Entrapment of microparticles within PDMS micropateterned patches to drug delivery applications

As proof-of-concepet, PCL microparticles were entrapped within PDMS micropatterning patches. (A–C) Entrapment effectiveness of microparticles with different diameters:
40 μm (A), 80 μm (B), and 160 μm (C), entrapped within the patches with varying micropillar spacings (40, 80, and 160 μm)

(D) In vitro cumulative release of tetracycline from the
patches at pH 5.5 and pH 7.4 at 37 °C in PBS.

(E) Antimicrobial activity of the different combinations: patches with  tetracycline-loaded alginate microparticles or bare tetracycline powder against two microorganisms-  gram-negative E. coli and gram-positive S. aureus.

Keywords: A-06 a - Biomaterials for drug delivery, A-07 r - Wound healing and tissue adhesives, A-07 b - Antibacterial
5:30 p.m. IX-OS35-04

A pH-responsive nanoparticle for functional miR-199a/b-3p delivery in hepatocellular carcinoma (#1009)

S. Shao1, Q. Hu1, W. Wu1, 2, T. Liang1, G. Tang2

1 The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Hepatobiliary and Pancreatic Surgery, Hangzhou, China
2 Zhejiang University, Department of Chemistry, Hangzhou, China

Introduction

Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the fourth leading cause of cancer death worldwide in 2018 [1]. Aberrant expression of key miRNAs in HCC significantly correlates with tumor metastasis and recurrence [2]. The level of miR-199a/b-3p, the third most abundant miRNA in human liver, is consistently decreased in HCC [3]. Moreover, it was proved to be a promising therapeutic target for HCC by regulating various cellular processes [3, 4]. By taking advantage of the slight difference of pH between normal tissues (~7.4) and extracellular environment of solid tumours (6.5-7.2) [5], here we designed a pH-responsive drug-delivery system to fulfill enhanced miRNA therapy in HCC.

Experimental Methods

We first synthesized a triblock copolymer of poly(ethylene glycol) (PEG) and admantyl modules bridged by a pH-labile linkage (CDM). Polyethylenimine-crosslinked β-cyclodextrins (PC) was synthesized according to the reported methods [6]. The complex PEG-CDM-PC was self-assembled from polycations and admantyl modules. The investigation of pH-responsive ability was completed with RNA release assay and diameter analysis under acidic environment. The therapeutic effect was demonstrated by in vitro studies up to now.

Results and Discussion

The complex was characterized by TEM and 1H-NMR (Fig. 1b, c). PEG-CDM-PC condensed miR-199a/b-3p had an average size of ~150nm and a zeta potential of ~+18.9mV. The elevated release of RNA from system and decreasing diameters of particles in solution with pH 6.5 demonstrated excellent pH-responsive effect of PEG-CDM-PC (Fig. 1d, e). PEG-CDM-PC pretreated with acid solution showed an enhanced cellular uptake (Fig. 2a) and efficient delivery of miR-199a/b-3p in Huh-7 cells (Fig. 2b). The increased miR-199a/b-3p suppressed tumor cell proliferation (Fig. 2c) by inhibiting mTOR, Bcl-2 and PAK4/Raf/MEK/ERK pathway (Fig. 2d). We are looking forward to positive in vivo study results which could confirm the preferential accumulation in cancer site and anti-proliferation effect in bearing tumour.

Conclusion

Our designed pH-responsive, self-assembled PEG-CDM-PC/miR-199a/b-3p is a promising therapeutic agent in future adjuvant therapy for HCC treatment.

References

1. F. Bray et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians 68, 394-424 (2018).

2. N. Yang, N. R. Ekanem, C. A. Sakyi, S. D. Ray, Hepatocellular carcinoma and microRNA: new perspectives on therapeutics and diagnostics. Advanced drug delivery reviews 81, 62-74 (2015).

3. J. Hou et al., Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma. Cancer cell 19, 232-243 (2011).

4. E. Callegari et al., miR-199a-3p Modulates MTOR and PAK4 Pathways and Inhibits Tumor Growth in a Hepatocellular Carcinoma Transgenic Mouse Model. Molecular therapy. Nucleic acids 11, 485-493 (2018).

5. S. Mura, J. Nicolas, P. Couvreur, Stimuli-responsive nanocarriers for drug delivery. Nature materials 12, 991-1003 (2013).

6. Q. Hu et al., A redox-sensitive, oligopeptide-guided, self-assembling, and efficiency-enhanced (ROSE) system for functional delivery of microRNA therapeutics for treatment of hepatocellular carcinoma. Biomaterials 104, 192-200 (2016).

Acknowledgement

This work was financially supported by the National Natural Science Foundation of China (grant number 81830089, 81472212 and 81502026) and the Zhejiang Provincial Natural Science Foundation (grant numbers LQ16H180002, LY18H160026).

Characterization of siRNA-encapsulated PEG-CDM-PC.
a) Scheme illustration showing preparation of PEG-CDM-Ad, PC and PEG-CDM-PC. b) Transmission electron microscopic (TEM) images of PEG-CD-PC, scale bar = 100nm. c) 1H-NMR characterization of PEG-CDM-PC and PEG-PC. The peak at δ 1.8-2.0 ppm characterizing CDM is highlighted in light green. d) Cumulative release of negative control RNA from the siRNA-encapsulated PEG-CDM-PC or PEG-PC in PBS with pH 6.5/7.4. e) The diameters of PEG-CDM-PC at various incubation times from 0-24 h.
PEG-CDM-PC-mediated cellular uptake and transfection of miR-199a/b-3p.
 a) Distribution of fluorescence intensity of Huh-7 cells after 4 h incubation with PBS, naked siRNA, PEG-CDM-PC/siRNA pretreated with pH 6.5 and 7.4, PEG-PC/siRNA and Lipo3000/siRNA. b) Dose-dependent cell viability after 72 h treatment with PEG-CDM-PC/miR-199a/b-3p pretreated with pH 6.5/7.4 and PEG-CDM-CD/NC. c) RT-PCR analysis of miR-199a/b-3p levels in Huh-7 cells. miR-199a/b-3p expression is normalized against PBS treatment. d) Expression of Bcl-2 and PAK4/Raf/MEK/ERK pathway by Western Blotting treated with PBS, PEG-CDM-PC/miR-199a/b-3p (pH 6.5/7.4) and PEG-CDM-CD/NC in Huh-7 cells. CD = PEG-CDM-PC/miR-199a/b-3p; non/mi = PEG-CD/miR-199a/b-3p; NC=negative control siRNA.
Keywords: A-06 d - Biomaterials for gene therapy, A-07 i - Kidney; liver and pancreas, A-08 b - Biodegradation
5:45 p.m. IX-OS35-05

Polydopamine-coated Zein-curcumin nanoparticles for targeted therapy of glioblastoma (#833)

H. Zhang1, I. S. Zuhorn1

1 University of Groningen, Biemedical Engineering, Groningen, Netherlands

Introduction

Effective treatment for Glioblastoma is severely limited by rapid resistance to agent therapies and the presence of the blood-brain barrier (BBB) that prevents therapeutics from reaching the brain. These issues can be addressed by the application of nanoparticles (NPs) that are effectively targeted and subsequently engage in transcytosis across the BBB.

In recent years, self-assembled biodegradable NPs from natural polymers, such as protein-based polymers, have attracted remarkable attention as potential drug delivery carriers. Zein, an alcohol-soluble protein, extracted from corn, has emerged as an ideal drug delivery system because of its intrinsic excellent biocompatible and biodegradable properties1. Compared with other proteins, zein has been extensively investigated for the encapsulation of bioactive compounds because of its unique capabilities, i.e., easy self-assembly into NPs, biocompatibility, and sustained drug release capability.

The one-step coating method for polydopamine (PDA) based on the oxidative self-polymerization of dopamine monomer in a week alkaline condition (pH 8.0-8.5) has aroused great interest in surface modification of biomaterials. The PDA layer on the surface of drug carriers greatly improves their hydrophilicity, colloidal stability, and reactivity with nucleophilic compounds for further modification via Michael addition or Schiff base reactions2.

Curcumin (CUR) is a polyphenol that is widely used in medicine for its pleiotropic anti-inflammatory, antimicrobial and anticancer activities3. Herein we report the design and synthesis of a dodecamer peptide (G23)-functionalized PDA- coated curcumin-loaded zein nanoparticle to traverse the blood-brain barrier (BBB) and deliver curcumin to glioblastoma cells.

Experimental Methods

The preparation of curcumin-loaded PDA-coated zein nanoparticles (Z-C-pDs) is based on a modified method of phase separation. Briefly, zein and curcumin (CUR) were dissolved in ethanol (80% v/v) at different weight ratios. Subsequently, 1ml of the solution was rapidly dispersed into 19 mL dopamine hydrochloride solution in Tris buffer (10mM, pH 8.5) for 12h at room temperature while stirring. The Z-C-pDs were collected by centrifugation and redispersed with deionized water. The same procedure was followed without adding curcumin to prepare ‘empty’ PDA-coated zein nanoparticles (Z-pDs). For the functionalization, Z-C-pDs were resuspended in Tris buffer (10mM, pH 7.4), which contained G23 peptide. After 2h stirring at room temperature, particles were collected by centrifugation and washed with deionized water. DLS was used to evaluate polydispersity and size. The morphology of the nanoparticles was demonstrated by AFM.  The cytotoxicity of the nanocomplexes was evaluated in human cerebrovascular endothelial (hCMEC/D3) cells and C6 glioma cells by fluorescence microscopy and MTT assays.

Results and Discussion

Z-C-pDs of 110 nm in size and with low polydispersity (PDI) were prepared at a zein and CUR weight ratio of 5:1. The NPs were spherical and showed a CUR loading efficacy of 8.1%. The surface charge was -37 mV, which provided greater colloidal stability than the positively charged CUR-loaded zein nanoparticles (Z-Cs) without PDA coating. Confocal laser scanning microscopy (CLSM) studies showed that the Z-C-pDs could effectively transport encapsulated curcumin into C6 glioma cells. Moreover, in vitro viability studies demonstrated that Z-C-pDs induced concentration-dependent cytotoxicity in C6 glioma cells, which was more efficient than with free CUR.

Conclusion

Polydopamine-coated zein-curcumin nanoparticles are able to induce cytotoxicity in C6 glioma cells and seem promising for the treatment of glioblastoma. The effect of the functionalization of these nanoparticles with the G23 peptide on their transport across the BBB needs to be determined, as well as its effect on cytotoxicity induction in C6 glioma.  

References

1.    Xue, J. et al. Zein-caseinate composite nanoparticles for bioactive delivery using curcumin as a probe compound. Food Hydrocoll. 83, 25–35 (2018).

2.    Park, J. et al. Polydopamine-based simple and versatile surface modification of polymeric nano drug carriers. ACS Nano 8, 3347–3356 (2014).

3.    Cheng, K. K. et al. Curcumin-conjugated magnetic nanoparticles for detecting amyloid plaques in Alzheimer’s disease mice using magnetic resonance imaging (MRI). Biomaterials 44, 155–172 (2015).

Acknowledgement

This work was supported by the Ministry of Economic Affairs, Netherlands) and the Dutch Ministry of Education, Culture and Science (Gravitation program 024.001.035).

Cellular cytotoxicity

Relative viability of C6 cells after incubation at different concentration of Z-pD, free Curcumin and Z-C-pD. The cell viability assessed by MTT assay after incubation for 24h.

Cellular Uptake of Curcumin

CLSM images of C6 Glioma cells after incubation with free Curcumin and Z-C-pD for 0.5h, 2h and 4h. Blue and green colors represent DAPI stained nuclear and curcumin fluorescence, respectively. The Scale bars represent 20 μm.

Keywords: A-06 a - Biomaterials for drug delivery, A-08 a - Biocompatibility, A-08 b - Biodegradation
6:00 p.m. IX-OS35-06

A photocurable and degradable polyester for nitric oxide release (#494)

M. F. de Oliveira1, M. G. de Oliveira1

1 University of Campinas, Institute of Chemistry, Campinas, Brazil

Introduction

Polyesters have been widely used as absorbable biomaterials. More recently, photocrosslinkable polyesters have emerged as a platform for the manufacturing of entirely absorbable implantable devices, such as expandable intracoronary stents.1 Photocrosslinkable materials allow the production of devices using photoinduced 3D printing technologies. Photocrosslinking has been achieved through the incorporation of pendent vinylic groups in the polymer backbone. One of the polyester with such properties is the methacrylated poly(dodecanediol citrate)(mPDC) (Fig. 1A), which undergoes photocrosslinking in the presence of a photoinitiator after irradiation with visible light. The potential biomedical application of mPDC may be greatly increased via the incorporation of drugs capable of improving its biocompatibility. One of the potential strategies for this propose is the incorporation of nitric oxide (NO) donors in the polymeric matrix.2 NO is an endogenous species responsible for endothelial regeneration, prevention of platelet adhesion and aggregation, among other physiological functions.3 Therefore, NO-releasing mPDC have the potential for production of absorbable blood-contacting medical devices. In this work, we developed a method for incorporating a NO donor into mPDC and demonstrate that this material is capable of releasing NO spontaneously after immersion in physiological medium.

Experimental Methods

The mPDC was synthesized via polycondensation of 1,12-dodecanediol and citric acid, followed by methacrylation.1 The polymer was characterized with Gel Permeation Chromatography (GPC), NMR and FTIR. mPDC was mixed with a photoinitiator (Irgacure 819) and ethanol to make a photocurable resin. The resin was cured with a visible light LED in a nitrogen purged environment to make the crosslinked materials. The NO-releasing ability of the polymer was introduced through the incorporation of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) (Fig. 1B). Scanning electron microscopy/Energy-dispersive X-ray spectroscopy (EDS) was used to confirm the incorporation of SNAP into the bulk of the polymer. The NO-release was measured by chemiluminescence using a Nitric Oxide Analyzer (NOA Sievers GE® model 280i) after the immersion of the polymer in PBS solution, pH 7.4 at 37°C, for crosslinked mPDC with different SNAP charges. The degradation of the crosslinked material was studied under accelerated condition, by measuring its mass loss in PBS solution at 60°C.

Results and Discussion

mPDC formation (Mw 1850 Da.) was characterized by NMR. FTIR spectroscopy confirmed the photocrosslinking of mPDC though the vanishing of the vinylic group at 1639 cm-1. The observed mass loss during accelerated degradation test showed that the polymers degrades at the rate of 0.15 wt% per day. SNAP incorporation into mPDC was confirmed through the observation of the characteristic peak of sulphur atoms in the EDS spectrum of mPDC/SNAP.

Real-time NO release measurements showed that, after immersion in PBS solution at 37°C, mPDC/SNAP releases NO at rates varying from 100 to 280 nmol g-1 min-1. Therefore, modulation of this rate by using different SNAP charges may allow for the inhibition of platelet adhesion, and thrombus formation. In addition, by changing the SNAP charge, different NO-release rates were obtained according to a linear dose-response (Fig. 2).

Conclusion

The mPDC/SNAP releases NO after immersion after aqueous medium. The rate of NO-release can be modulated by using different SNAP charges. These results indicate that mPDC/SNAP is a potential degradable polymeric material for local NO release from implantable medical devices.

References

1. R. Van Lith, et al. "3D‐Printing Strong High‐Resolution Antioxidant Bioresorbable Vascular Stents." Advanced Materials Technologies 1.9 (2016): 1600138.

2. V. Baldim, M. G. de Oliveira. Poly-ε-caprolactone/polysulphydrylated polyester blend: A platform for topical and degradable nitric oxide-releasing materials. European Polymer Journal, 109, 2018, 143-152.

3. C. Liang, et al. Biomimetic cardiovascular stents for in vivo re-endothelialization. Biomaterials, v. 103, p. 170-182, 2016.

Acknowledgement

MFO received a studentship from the São Paulo Research Foundation (FAPESP) (process 2018/02520-5). MGO acknowledges FAPESP for financial support (process 2016/02414-5).

Structure of mPDC and SNAP NO-release reaction

Fig. 1. (A) Structure of methacrylated poly(dodecanediol citrate). (B) NO-release reaction of S-nitroso-N-acetylpenicillamine (SNAP) incorporated into mPDC matrix.

NO release profiles of mPDC/SNAP
Real time NO-release curves obtained from SNAP incorporated into mPDC at three different concentrations. Inset: Dose-response of NO release rate.
Keywords: A-07 q - Vascular grafts incl. stents, A-01 f - Polymeric biomaterials, A-06 a - Biomaterials for drug delivery