BIOMATERIALS FOR CELL

AND DRUG DELIVERY 2019

Workshop 17th -19th September 2019

Metropolitan University, Manchester, UK

Workshop Venue: Geoffrey Manton, Room GM230, Manchester Metropolitan

University

Hotel Pendulum Address: Sackville St, Manchester M1 3BB

Tuesday 17th September 2019

09.30 – 10.30 COFFEE & REGISTRATION

10.20 – 10.30 Welcome, Aims and Expectations

SESSION 1 Chairs: Anastasiya Solovieva & Olga Efremova

10.30 – 11.10 Veniamin Khazanov “Biomaterials for Drug Delivery and Development”

(Keynote)

11.10 – 11.30 Lisa White “ECM Biomaterials”

11.30 – 11.50 Jin Ding “The economic impact of 3D printing on pharmaceutical

industry”

11.50 – 12.10 Qifeng Qian “Industrialisation of 3D inkjet printing for the manufacture

of solid dosage forms”

12.10 – 12.30 Maria Smetanina “Quantitative and structural characteristics of

mitochondrial DNA in varicose veins of lower extremities”

LUNCH

SESSION 2 Chairs: Veniamin Khazanov & Timothy Douglas

13.40 – 14.00 Ales Buyakov “Personalized approach to the cancer patients bone

defects reconstruction with porous bioactive ceramic implants”

14.00 – 14.20 Natalia Porfiryeva “Interpolyelectrolyte complexes as formulations for

drug delivery to the brain”

14.20- 14.40 Mareike Posner “Macromolecular protein complexes for biomedical and

biotechnological applications”

14.40 – 15.00 Anton Popov “Ceria nanoparticles-decorated micro- and nanocarriers for

cellular drug delivery and theranostic application”

15.00 – 15.20 Evgeny Apartsin “Smart amphiphilic dendrimer vesicles for drug

delivery”

15.20 – 15.40 Pavel Gershkovich “Lipid-based drug delivery to facilitate lymphatic

transport and to increase systemic bioavailability”

15.40 – 16.00 Mohamed Elsawy “Molecular control of drug release from peptide

nanogels”

COFFEE BREAK

SESSION 3 Chairs: Anton Popov & Mareike Posner

16.30 – 17.00 Zakhar Popov “Plasma polymerized films for biomolecular

immobilization from theoretical modelling and experimental study”

(Keynote)

17.00 – 17.20 Sergey Ankov “Nanoaerosol forms of nonsteroidal anti-inflammatory

and anti-tuberculosis medicinal substances”

17.20 – 17.40 Iwan Palme “Use of Electron Beam Treatment to Control Release of

Therapeutic Agents from Absorbable Polymer Medical Devices”

17.40 – 18.00 Anastasiia Kotlyarova “Porous carriers based on aluminum oxide and

polymethylsiloxane polyhydrate for controlled/modulated drug delivery”

18.00 – 18.20 Maria Lomova Methods of submicron and micron drug delivery carriers

investigation in vivo and in vitro

19.30 – 21.30 Welcome DINNER @ Hotel Pendulum

Wednesday 18th September 2019

SESSION 1 Chairs: Yuliya Laricheva & Iwan Palme

9.30 – 10:00 Anastasiya Solovieva “Modification of PCL nanofibers for a wide range

of tasks in regenerative medicine” (Keynote)

10:00 – 10:20 Chia-Chen Hsu “Culture of Human Induced Pluripotent Stem Cell-

Derived Neurons in Multi-phase Granular Hydrogels”

10.20 – 10.40 Timothy Douglas “Dairy-derived biomaterials: hydrogels and implant

coatings”

10.40 – 11.00 Richard Balint Cardiac Tissue Engineering Using Graphene-Polymer

Composites

COFFEE BREAK

SESSION 2 Chairs: Zakhar Popov & Masoomeh Bazzar

11.30 – 11.50 Farshid Sefat “Biodegradable Scaffolds for Vascular Tissue

Engineering”

11.50 – 12.10 Dmitry Bagrov “The structure of electrospun mats made of polylactide

and bovine serum albumin”

12:10 – 12.30 Phillip Chivers ”Shining a Light on Stem Cell Differentiation: Photo-

patterned Supramolecular Hydrogels for Tissue Engineering

LUNCH

SESSION 3 Chairs: Alessandro Faroni & Tatiana Pozmogova

13.30 – 14.00 Araida Hidalgo “2D vs 3D Models for Drug Toxicology” (Keynote)

14.00 – 14.20 Ling Yong Xin “Identification of Antifungal Copolymer for Biomedical

Devices”

14.20 – 14.40 Zhan Yuin Ong “Bioinspired Design of Multifunctional Nanomaterials

for Anti-cancer and Anti-microbial Applications”

14.40 – 15.00 Alexey Chubarov “Multifunctional human serum albumin or

polyethyleneimine therapeutic nucleotide conjugates as a potential

theranostic agents”

15.00 – 15.20 Nadezhda Dyrkheeva “Development of the Tdp 1 inhibitors as drug

precursors”

15.20 – 15.40 Masoomeh Bazzar “Encapsulated near infrared polymeric "nano

particles" a potential for theranostic nanomedicine applications”

15.40 – 16.00 Alessandro Faroni “Self‐Assembling Peptide Hydrogel Matrices

Improve the Neurotrophic Potential of Human Adipose‐Derived Stem

Cells”

16.00 – 17.30 Networking: Speed dating & COFFEE

18.00 – 21.00 Workshop DINNER @ Albert’s Chop House (Manchester city

centre)

Thursday 19th September 2019

SESSION 1 Chairs: Maria Smetanina & Richard Balint

09.00 – 09.30 Olga Efremova “Octahedral clusters – versatile materials for biomedical

applications” (Keynote)

09.30 – 09.50 Yuliya Laricheva “Interaction of hydrophobic trinuclear tungsten

clusters with phospholipid bilayers: a route towards functional hybrid

materials”

09.50 – 10.10 Tatiana Pozmogova “Luminescent silica microparticles as new

detectable agents for protein transduction”

10.10 – 10. 30 Aron Teklemariam TBC

COFFEE BREAK

SESSION 2 Chairs: Olga Efremova & Araida Hidalgo & Anastasiya Solovieva

10.30 – 11.40 Sandpit on joint research applications between UK and Russian

researchers

11.40 – 13.00 Feedback session and legacy action plan

12.00 – 12.15 Closing Remarks

LUNCH & End of workshop

Culture of Human Induced Pluripotent Stem Cell-Derived Neurons in Multi-phase Granular

Hydrogels

Chia-Chen Hsu1, Julian H George1, Sharlayne Waller1, Cyril Besnard2, Alexander M. Korsunsky2, Hua Ye1,2,

Zhanfeng Cui1,2

1Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, OX3 7DQ, UK

2Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK

Hydrogels as a supporting substrate to facilitate cell transplantation and tissue engineering applications are

promising materials for neural repair and regeneration due to its injectability and ideal mechanical properties

resembling the nature nervous system. It has been shown that hydrogels capable of dynamically encompassing

and exchanging external stimuli, including extracellular matrices, soluble factors and cells can bridge across

damaged neural tissues and aid in repair. Nevertheless, a bulk hydrogel consisting of dense polymer networks

without a perfusion system often leads to suboptimal microenvironments which impede nutrient exchanges and

cell-cell interactions.

Herein, we developed a multiphase hydrogel system composed of granular hyaluronic acid (HA) hydrogels using

a designed 3D printed toolset. By creating hierarchical structures within a hydrogel, it allows better recapitulation

of the native tissue utilising granular microgels as the structural support and secondary interphase rich in

extracellular matrices to encapsulate cells and soluble factors. The results show the diameter of hydrogel

granules is ~2 times the diameter of the selected nylon mesh used for granulation. The gel formation was

completed after ~30 minutes adding the secondary crosslinker (10%, 20%, 30%, 50%) and their storage moduli

measured at the frequency of 10 Hz range from 100-200 Pa, which has been previously shown to promote

neurogenic differentiation. The HA granular hydrogels could provide longer-term support for clinically relevant

human induced pluripotent stem cell-derived neural stem cells compared to the bulk hydrogels. While there was

no significant difference in cell viability on Day 1, cell viability was significantly higher in the granular hydrogels

(60.4 ± 6.0 %, 62.9 ± 7.0 %) compared to the bulk hydrogels (40.3 ± 4.8 %, 35.7 ± 6.1 %) on Day 3 and Day 7.

Furthermore, the granular hydrogels can promote structural maturation in neurons on Day 7 with more neurite

bearing cells (>10 cells per analysed fields) and longer neurite extension (87.9 ± 7.7 µm) compared to the bulk

hydrogels (≤2 cells per analysed fields; 32.0 ± 26.3 µm). Through the enhancement of cell support and neurite

outgrowth, the developed hydrogels present a valuable platform for stem cell-based therapy for neural repair

and regeneration.

The economic impact of 3D printing on pharmaceutical industry

Jin Ding

Centre for Additive Manufacturing, University of Nottingham

The challenges faced by the pharmaceutical sector include the innovation of customized medication

and the increasing cost of medications. This is exacerbated by rapid progress in the fields of genomics

and diagnostics. At the same time, the underlying conventional batch manufacturing processes

routinely used to manufacture medicines have remained unchanged for nearly a century. With the

ability of producing on-demand, personalized and complex products, Additive Manufacturing (AM)

promises a pathway towards more precise drugs and improved medication adherence while at the

same time facilitating a reorientation towards smaller-batch size and on-demand manufacturing.

The adoption of 3D printing (3DP), also known as additive manufacturing, has been heralded as a new

way of manufacturing in many industries, including the pharmaceutical sector. Engineers, managers

and technology observers, however, are still struggling to understand 3DP’s potential to deliver value

in manufacturing pharmaceuticals. This research addresses this gap in knowledge by exploring the

practical operation of manufacturing solid dosage forms (i.e. pills) from a cost perspective. Specifically,

this paper develops a state-of-the-art 3DP cost model of the detailed-analysis type including the

aspects of capacity utilisation, integration with necessary ancillary process elements, build failure and

product rejection. The parameters of this cost model are estimated in an exploratory fashion through

a run of build experiments involving a material jetting system to fabricate solid dosage forms

containing Ropinirole, a medication developed to treat Parkinson’s disease.

We develop a robust cost model for ink-jetted solid dosage forms by incorporating factors of batch

size, necessary ancillary process elements, process failure, product rejection and material waste

streams. The experimental data provides detailed insight into the relationships and between

technological parameters and cost characteristics. The results show that the manufacturing cost and

the build time can be cut by upsizing the nozzles or building more print heads. Hence, material jetting

techniques enable easy scale-up, it potentially offers a fast and flexible approach for fabricating drugs

for niche market. This is of great significance for the pharmaceutical industry in the coming age of

customization.

Biodegradable Scaffolds for Vascular Tissue Engineering

M. Bazgir1, W. Zhang2, M. Katsikogianni3, M. Youseffi1, F. Sefat1,4

1 Faculty of Engineering and Informatics, Department of Biomedical and Electronics Engineering, University of Bradford, Bradford, UK

2 State Key Laboratory of Polymer Materials Engineering, Polymer Research Institute, Sichuan University, Chengdu, China

3 Faculty of Life Science, Department of Chemistry, University of Bradford, Bradford, UK

4 Interdisciplinary Research Centre in Polymer Science & Technology (IRC Polymer), University of Bradford, Bradford, UK

* Corresponding Author: F.Sefat1@Bradford.ac.uk

Annually, ~ 80,000 people die in the United Kingdom due to myocardial infarction, congestive heart failure, stroke, and vascular diseases. Currently, the gold standard treatment for blood vessel disorders is using autografts, for instance internal mammary artery (IMA) graft, or saphenous vein graft (SVG). There are some limitations in regards to this type of treatment such as lack of donor site, as well as patient conditions lead to problems associated with optimum transplantation. Therefore, the main aim of this work is to fabricate a synthetic blood vessel that mimics the natural arteries. Biomaterials intended to be used for this purpose have to be biodegradable with optimum mechanical properties and significant cell proliferation.

For this work, Poly (ε-caprolactone) (PCL) and poly (lactide-co-glycolic-acid) or PLGA have been used to fabricate a hollow scaffold. Electrospinning technique was employed to fabricate a suitable tubular scaffold and hence four tubular shape vascular grafts were fabricated with diameter of 6mm and each tube was designed differently. The above-mentioned biodegradable polymers were successfully fabricated into tubular vessels and their morphology have been investigated by scanning electron microscopy (SEM). Furthermore, the biodegradability properties of both polymers were also studied over a period of 12 weeks at room temperature. The scaffolds were exposed to a controlled temperature of 37˚C for the duration of four weeks. It was found that the morphology of the electrospun scaffolds showed similarity to the layers of the native blood vessel. Overall, all four scaffolds displayed exceptional fibre structure and excellent degradability over the course of the experiment. The percentage weight-loss of the scaffold increased further confirming useful degradability of the scaffolds. Although, both PCL and PLGA degraded, the results indicated that PCL scaffolds had greater degradability than PLGA scaffolds. The mechanical properties were investigated with the tensile testing machine which showed that the scaffolds produced by the coaxial needle had the highest tensile strength (6.75 ±1.44 MPa), % elongation at fracture 141.78 ± 45.01 % and Young’s modulus 17.36 ± 8.28 MPa.

Macromolecular protein complexes for biomedical and biotechnological applications

Mareike Posner

Manchester Metropolitan University, UK

Here, I describe the use of a 2-oxoacid dehydrogenase complex (2-OADHC) from the Archaeon Thermoplasma acidophilum in graphene-based biosensing. The Thermoplasma complex belongs to the superfamily of 2-OADHCs, which represent some of the largest protein complexes known in nature. Members of the 2-OADHC superfamily have previously been used in other biotechnology applications such as phage display. Their self-assembly into cage-like structures has potential in therapeutic delivery. Like other members of this family, the Thermoplasma 2-OADHC has a modular design, making it amenable to customisation e.g. display of functional groups/proteins. In addition, the Thermoplasma complex is thermostable and its assembly is driven by temperature. This work adds to the ongoing efforts in graphene biofunctionalization by exploring the potential of macromolecular protein complexes.

Self‐Assembling Peptide Hydrogel Matrices Improve the Neurotrophic Potential of Human Adipose‐Derived

Stem Cells

Dr Alessandro Faroni

Faculty of Biology Medicine & Health, School of Biological Sciences, Division of Cell Matrix Biology and

Regenerative Medicine, The University of Manchester

Despite advances in microsurgical techniques, treatment options to restore prior function following major

peripheral nerve injury remain unavailable. In the presence of a nerve gap, autologous nerve grafting remains

the therapy of choice. Much recent experimental work has focused on the development of artificial constructs

incorporating novel smart biomaterials and stem cells, aspiring to match and improve the outcomes of nerve

autografting. Human Adipose-derived Stem Cells (dhASC) can be chemically stimulated in vitro to produce

essential growth factors able to improve nerve regeneration outcomes; however, these properties are lost when

the chemical stimulation is withdrawn and survival rate upon transplantation is low. Our aim was to exploit the

properties of novel, fully synthetic hydrogel matrices to retain and improve the neurotrophic characteristics of

dhASC, with a view to delivering stem cell therapies for nerve repair. dhASC were cultured on PeptiGel®-Alpha 1

and PeptiGel®-Alpha 2 self-assembling peptide hydrogels showing comparable viability to Collagen I gels used as

controls. Alpha 2 substrates allowed long-term dhASC cultures (up to 3 weeks), and retained mechanical

properties comparable to peripheral nerve tissues. Culturing dhASC on Alpha 1 and Alpha 2 substrates allowed

the maintenance of neurotrophic features, such as the expression of growth factors and several neuroglial

markers. Both Alpha 1 and Alpha 2 substrates were suitable for the culture of peripheral sensory neurons,

permitting the sprouting of neuronal extensions without the need of biological extracellular matrices, and

preserving neuronal function. We propose Alpha 1 and Alpha 2 substrates loaded with hdASC as promising

candidates for the development of tissue engineering therapies for the repair of peripheral nerve injuries.

Identification of Antifungal Copolymer for Biomedical Devices

Ling Yong Xin

University of Nottingham, UK

Silicone rubber (Polydimethylsiloxane, PDMS) is commonly used for production of medical devices as it has excellent biocompatibility, flexibility, processability and chemical resistance. However, in many of its biomedical applications, PDMS can attract yeast such as Candida albicans leading to fungal infections. Till date, fungal infections from medical-assisted device caused by Candida albicans alone has resulted in up to 30% mortality rate. Incorporation of antifungal polymer would minimize both complication in patient’s health from frequent changing of medical devices and would also reduce the mortality rate caused by such infections. In this work, high throughput methodologies were used to screen large number of methacrylate and acrylate identifying monomers that can either be use for dip coating or additive manufacturing of medical device. Following the evaluation, we chose 10 monomers to be mix at different ratio in order to obtain copolymers with improved viscosity, reactivity and mechanical properties. This instantly widens the option of polymeric materials available for biomedical industry.

Use of Electron Beam Treatment to Control Release of Therapeutic Agents from

Absorbable Polymer Medical Devices

Iwan Palmer1, Susan Clarke2, Fraser Buchanan3

1School of Pharmacy & Pharmaceutical Sciences, Cardiff University; 2School of Mechanical & Aerospace Engineering, Queen’s University Belfast; 3School of Nursing & Midwifery, Queen’s University Belfast

Introduction

Electron beam (ebeam) treatment can be used to control the degradation of, and release of incorporated therapeutic agents from, absorbable polymer medical devices1. Absorbable polymers have potential in the biomedical device field due to their tailorable material properties. However, setbacks such as the 2007 recall of the CALAXO® interference screw2, significantly slowed the adoption of this technology. A key factor in improving implant performance is the control of polymer degradation. Bulk degradation is typically observed in commonly used absorbable polymers3 resulting in early loss of mechanical integrity and an inflammatory acid burst effect. ebeam treatment can be used to control polymer degradation. It involves electron beam irradiation and results in decreased average molecular weight at the implant surface through chain scission4. The extent of surface degradation can be controlled by adjusting the accelerating voltage. This allows degradation to proceed from the outside of the polymer towards the centre, resulting in early stage mass loss and therapeutic release while maintaining internal mechanical strength.

Materials & Methods

Commercial orthopaedic screws formulated from a blend of poly(L-lactide-co-glycolide) (PLGA) and prototype screws of the same formulation with additional 10 wt% β-tricalcium phosphate (β-TCP) (PLGA-TCP) underwent ebeam treatment. A surface dosage of 500 kGy and an accelerating voltage of 115 keV (COMET Group) were used bilaterally, giving a penetration depth of approx. 50 μm around the screws’ thread. Treated and untreated screws underwent 36 weeks in vitro degradation in phosphate buffered saline (PBS) at 37 °C with mass loss, pH change and Ca release recorded. Implants were also investigated in vivo using a rabbit model. Screws were implanted bilaterally into distal femoral condyle defects and retrieved at 12 and 36 weeks. Bone growth and osteoclast activity were assessed using toluidine blue and tartrate-resistant acid phosphatase (TRAP) staining respectively.

Results & Discussion

In Vitro: No mass change was observed after 4 weeks, but after 12 weeks mass was significantly reduced for treated samples. ebeam treatment resulted in significantly greater mass loss in both PLGA and the PLGA-TCP screws at 12 and 36 weeks. ebeam treatment also accelerated the drop in pH of PBS containing PLGA samples over 10 weeks, while the pH of untreated samples remained relatively constant. Difference in pH between treated and untreated PLGA samples continued to increase up to around 22 weeks. A similar trend was seen in Ca containing samples; marked differences in Ca release were observed between treated and untreated PLGA-TCP samples. After 2 weeks, released Ca was similar for untreated and treated samples, however between weeks 4 and 24, significantly more Ca was released from ebeam treated samples. At each timepoint between 4 and 24 weeks, Ca release increased by over 100 % following treatment. From 28 weeks onwards, treatment effect on Ca release was less marked. This confirms that ebeam treatment accelerates surface degradation of the polymer.

In Vivo: ebeam treatment did not adversely affect bone growth or osteoclast activity around implants. Extensive bone growth was observed around the perimeter of all implant types along with limited osteoclastic activity. By 36 weeks fragmentation was observed along with bone growth into fissures. Fissures, and associated bone ingrowth, were equally extensive for treated screws, suggesting that accelerated surface degradation does not compromise in vivo biocompatibility.

Conclusions

ebeam treatment allows early surface degradation of absorbable polymer devices. Pre-degradation of device surfaces in this way can significantly increase the early release rate of incorporated therapeutic agents. This is

likely to be of particular value in orthopaedic devices as the greatest increase in release is observed at time points associated with typical healing times of common fractures.

References

1. Palmer, I. et al. (2019) Bone Jt. Res. 8, 266–274.

2. Cox, C. L. et al. (2010) J. Surg. Orthop. Adv. 19, 121–124.

3. von Burkersroda, F. et al. (2002) Biomaterials 23, 4221–4231.

4. Leonard, D. J. et al. (2009) J. Biomed. Mater. Res. A 89A, 567–574.

Acknowledgements

The authors would like to acknowledge Invest Northern Ireland for funding this work (POC 410). Also, thanks to the COMET Group for their assistance with the electron beam treatment.

Lipid-based drug delivery to facilitate lymphatic transport and to increase systemic bioavailability

Dr Pavel Gershkovich

School of Pharmacy, University of Nottingham, UK

The lymphatic system represents a unidirectional flow network consisting of lymph vessels, lymph nodes and lymphoid tissues which serve to absorb the excess fluid and proteins, not reabsorbed by the blood capillaries, to the systemic circulation. Additional major functions of the lymphatic system include proliferation and maturation of immune cells, as well as absorption of dietary lipids. Lymphatic transport of drugs following oral administration has been shown to increase the bioavailability substantially in comparison to absorption pathways that do not involve the lymphatic system. The intestinal lymphatic transport of lipophilic drugs following oral administration is coupled with absorption of dietary lipids and has been of particular interest for pharmaceutical scientists in the last three decades. Importantly, drugs transported via the intestinal lymphatics bypass the liver and avoid hepatic first pass metabolism, which results in increased systemic bioavailability.

However, an issue that has been practically overlooked in the past is that the lymphatic system has functional importance, and drugs that are transported via this route can exert their activity within the lymphatic system itself. The concept of the lymphatic system as an effective compartment for drugs is novel and has an enormous potential for dramatically improving the treatment outcomes of a number of diseases with pathophysiology that is associated with the lymphatic system. Examples include autoimmune diseases, metastatic process and HIV infection. Targeting of drugs to the lymphatic system for treatment of these conditions has the potential to maximize their therapeutic benefits while minimizing the side effects associated with systemic exposure.

Over the last 5 years we have successfully delivered a number of relevant compounds to the intestinal lymphatic system. For example, the concentration of natural lipophilic cannabinoids delta-9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) was 250-fold higher in lymph compared to plasma in rats following oral administration with lipids. These high levels resulted in immunomodulatory activity that was not observed at lower levels of these compounds in plasma. These data demonstrate that targeting cannabinoids to the intestinal lymphatic system can be a promising approach for treatment of autoimmune diseases.

Another example is delivery of anticancer compound bexarotene to mesteneric lymph nodes for improved treatment of lymphomas. Although this compound does not have right physicochemical properties to be targeted to the lymphatics as it is – we have synthesised highly lipophilic prodrugs of bexarotene. Activated ester prodrug approach resulted in 17-fold higher concentrations of the drug in mesenteric lymph nodes compared to when the drug alone was administered orally.

Finally, we recently started to work with antiretroviral compounds. Gut-associated lymphoid tissues are one of the main reservoirs of the virus and is a substantial barrier to eradication of the disease. We have showed that lipophilic prodrug approach combined with lipid-based delivery is a promising approach to target antiretroviral drugs such as lopinavir and other protease inhibitors to gut-associated lymphoid tissues.

In conclusion, lipids facilitate lymphatic transport of lipophilic drugs following oral administration. This leads to increase in systemic bioavailability, and most importantly – to very high concentrations of drugs within the lymphatic system itself. In cases were drug is not sufficiently lipophilic for this delivery – lipophilic prodrugs have been demonstrated as promising approach. Lymphatic targeting has potential to improve treatment of multiple conditions including autoimmune diseases, cancer and HIV/AIDS.

Cardiac Tissue Engineering Using Graphene-Polymer Composites

R. Balint1, L. A. Hidalgo-Bastida2,3,4

: 1Manchester Institute of Biotechnology (MIB), University of Manchester, United Kingdom, 2Centre for

Biomedicine, 3Centre for Advanced Materials and Surface Engineering, 4Centre for Musculoskeletal Science and

Sports Medicine, Manchester Metropolitan University, UK

Abstract

Electroactive biomaterials are of interest for the engineering of excitable tissues and implantable devices. Metals,

such as gold and titanium, are commonly used, but can be expensive and suffer from mechanical incompatibility

with most tissue types. Intrinsically conductive polymers (e.g PPy, PEDOT) have been extensively investigated,

but are difficult to process once synthesised and also lose their conductivity (and often mechanical integrity) with

repeated cycles of electrical stimulation [1]. In this study, we aimed to explore whether compositing the highly

electrically conductive 2D material graphene with polymers could yield a material suitable for the engineering of

excitable tissue, with a focus on cardiac cells.

A custom bioreactor system was designed and built for effective electrical stimulation of cells cultured on a

biomaterial surface. FEM computer simulations were performed to assess the behaviour of the electrical field

and current inside the bioreactor chamber. Polycaprolactone (PCL) and polyurethane (PU) were composited with

four types of commercially available graphene at weight percentages ranging from 0 to 75%. These composites

were tested for electrical conductivity, wettability, mechanical properties and surface morphology.

Biocompatibility was tested with C2C12 mural myoblasts and RN22 rat Schwann cells. The composites’

performance was further explored with human cardiac progenitors and PSC-derived cardiomyocytes, with and

without fibronectin coating, and with and without 2 and 20 ms mono- and bi-phasic electrical stimulation.

Response was tested using PicoGreen, Vybrant DiD staining, qRT-PCR for 9 cardiac marker genes and superarrays.

Computer simulations determined 1.7 S/m as the critical electrical conductivity for the substrates. All four

graphene-polymer types were able to achieve conductivities exceeding this value. Conductivity was not

decreased after 6 days of continuous stimulation. It was found that addition of polycarboxylate-functionalised

graphene decreased contact angle to approx. 60°, while all other types increased it. Material roughness, porosity,

reduced modulus and hardness were also increased by the addition of graphene. C2C12 and RN22 cells adhered

and proliferated on hydrophilic graphene composites to an equivalent or greater extent than on pure polymers.

Fibronectin coated hydrophilic composites showed excellent performance with cardiac cells, reaching 5 times

greater cell numbers within only 5 days of in vitro culture. Electrical stimulation delivered through PU-graphene

substrates significantly increased the expression of key cardiac developmental genes GATA4, TBX5 and SMARCD3

in cardiac progenitors, and GATA4, NKX2-5, TBX5, SMARCD3 and GATA6 in PSC-derived cardiomyocytes. This

study demonstrates that graphene-polymer composites offer a valuable alternative to conductive polymers in

engineering of cardiac tissues.

Acknowledgements: We thank the EPSRC Doctoral Prize Fellowship and the EPSRC Postdoctoral Fellowship

(EP/P016898/ 1) funding for financial support.

References: [1] Balint R et al. Acta Biomater. 2014; 10:2341-53.

Dairy-derived biomaterials: hydrogels and implant coatings

Timothy E.L. Douglas

Engineering Department, Lancaster University

Whey protein Isolate (WPI) is a by-product in dairy industry which is known to promote cell growth when in

solution. Its main component β-lactoglobulin is able to:

- Form hydrogels upon heating, allowing sterilization by autoclaving. These hydrogels can support the growth of

bone-forming cells. By addition of a mineral phase, e.g. by enzymatic mineralization or addition of pre-formed

mineral particles, the mechanical properties, cell proliferation can be improved. By addition of further

biomolecules from the food industry, such as polyphenols, further properties such as antibacterial activity can

be imparted.

- assemble into fibrils with functional properties that can serve as a new coating material for surfaces such as

titanium alloy (Ti6Al4V), a commonly used material for load-bearing applications in orthopaedics. We have

developed a sterile fibrillar coating for Ti6Al4V with the ultimate aim of improving new bone formation at the

surface and better osseointegration and stability. Studies have shown the ability of the fibrillary coatings to

support bone-forming cell growth.

Industrialisation of 3D inkjet printing for the manufacture of solid dosage forms

Qifeng Qian

Added Scientific, UK

Many AM techniques have the potential to revolutionize manufacturing industry and inkjet printing is one such

method which can dispense liquid material in a form of tiny droplets (i.e. picolitre scale) through a printhead

orifice. The solidification of the deposited material is usually completed by chemical reaction or solvent

evaporation to form a 3D object in a layer-by-layer manner. Several functional ink formulations have been

developed for applications such as prototypes, electronics and medicines at University of Nottingham and a great

effort has been input for industrial development at Added Scientific. A recent translation from 3D inkjet printing

medicines to a scalable manufacturing approach for pharmaceutical industry has been explored where it shows

a great potential to enable the precise fabrication of oral dosage forms for personalized medicines. Using 3D

inkjet printing as a production tool to manufacture medicines have demonstrated a high design freedom of the

pills such that the dosage, sizes and shapes can be controlled. To enable the integration of inkjet printing into

pharmaceutical practice, it is necessary to further evaluate this novel technology under standard GMP conditions

which would lead to new challenges and opportunities.

Bioinspired Design of Multifunctional Nanomaterials for Anti-cancer and Anti-microbial Applications

Dr Zhan Yuin Ong

School of Physics & School of Medicine, University of Leeds, Z.Y.Ong@leeds.ac.uk

The ability to precisely tune and incorporate various functionalities onto nanoscale materials has offered

unprecedented opportunities for improving the treatment of human diseases. The large neutral amino acid-

transporter-1 (LAT-1), which transports large bulky amino acids is fast emerging as an attractive target for the

imaging and treatment of human cancers due to its overexpression and positive correlation with tumour

aggressiveness. Recently, we have reported the bioinspired design of highly uniform gold nano-urchins using the

LAT-1 substrate, L-dopa, and demonstrated how it opens exciting avenues for targeting fast-growing cancer cells

and the highly impermeable blood-brain barrier. The ease of tuning their optical properties for selective

photothermal ablation of triple negative breast cancer will be discussed. Lastly, with antimicrobial resistance

being a key unmet medical need, there is a need to develop novel strategies with alternative mechanisms of

action from small molecule antibiotics. Here, I will discuss the design of a novel hydrogel formulation

incorporating antimicrobial peptide (AMP)-encapsulated liposomes and gold nanoparticles. The application of

this multifunctional hydrogel formulation for the light-activated release of AMPs and enhanced photothermal

treatment of pathogenic bacteria will be discussed.

Nanoaerosol forms of nonsteroidal anti-inflammatory and anti-tuberculosis medicinal substances

Dr Sergey Ankov

N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry (NIOCH SB RAS), Laboratory of

Pharmacological Research, Russia

Modern therapeutic methods of treatment of various diseases are increasingly using aerosol means of delivery of medicines to the patient's lungs. This drug delivery method is used to treat respiratory diseases such as asthma, bronchitis, cystic fibrosis, bacterial or fungal infection, chronic obstructive pulmonary disease, primary pulmonary hypertension (Ruge et al., 2013). On the other hand, inhalation has great potential for the treatment of systemic diseases (Laube, 2005). The main advantages of inhalation delivery are the speed of achieving the drug effect, ease of administration, no need for medical personnel, no pain. In addition, metabolism in the lungs is manifested to a much lesser extent than in the gastrointestinal tract. In addition, aerosol delivery has no restrictions associated with the use of water-soluble drugs as opposed to injection therapy.

In the laboratory of nanoparticles Professor A. A. Onischuk was obtained aerosol formulations of ibuprofen, diclofenac sodium and isoniazid. For ibuprofen, the effectiveness of anti-inflammatory action in vivo tests on the model of "histamine inflammation" was evaluated, as well as pharmacokinetics in serum was established, and compared with the pharmacokinetics of oral delivery. For isoniazid shown pharmacokinetics aerosol form in serum and organs, as well as in: lungs, liver.

According to the results of the experiments, the effective dose of ibuprofen in aerosol form was reduced by more than 3000 times compared to oral. This was also confirmed by measurements of serum concentrations, depending on the time. Isoniazid showed comparable to oral pharmacokinetics, due to its high water solubility and high oral bioavailability.

1. Laube, B.L. (2005). The Expanding Role of Aerosols in Systemic Drug Delivery, Gene Therapy, and Vaccination.

Respiratory Care 50, 1161 - 1176.

3. Ruge, C.A., Kirch, J., & Lehr, C.-M. (2013). Pulmonary drug delivery: from generating aerosols to overcoming

biological barriers - therapeutic possibilities and technological challenges. The Lancet Respiratory Medicine, 1,

402–413.

Smart amphiphilic dendrimer vesicles for drug delivery

Evgeny K. Apartsin,a,b Valeria I. Arkhipova,a,b Javier Sánchez-Nieves,c,d Alya G. Venyaminova,a F. Javier de la Mata, c,d Rafael Gómez c,d

aInstitute of Chemical Biology and Fundamental Medicine SB RAS, 8, Lavrentiev ave., 630090

Novosibirsk, Russia. bDepartment of Natural Sciences, Novosibirsk State University, Novosibirsk, Russia.

cDepartamento de Quimica Organica y Quimica Inorganica, UAH-IQAR, Universidad de Alcala, 28805 Alcala de Henares, Spain.

dNetworking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain.

E-mail: eka@niboch.nsc.ru

Soft nanosized biomaterials hold great potential in nanomedicine as prospective drug carriers. In particular, supramolecular assemblies with controllable structure and properties (artificial micelles, vesicles) are highly promising due to their ability to increase the stability and bioavailability of low-molecular and macromolecular drug formulations. Dendrimers – symmetric hyperbranched polymers – have been recently introduced as building blocks for such assemblies. Herein, we report the design of pH-sensitive vesicles built of amphiphilic carbosilane dendrimers as well as their loading with anti-cancer chemodrugs and therapeutic nucleic acids. A series of amphiphilic dendrimers has been synthesized in this work. These amphiphilic dendrimers consist of three major elements: hydrophobic aliphatic tails forming a bilayer during the self-assembly; pH-sensitive triazine fragment driving vesicles assemblage/disassemblage; cationic carbosilane dendron acting as hydrophilic head and defining the morphology of the nanoparticles and responsible for the interactions with the cell surface. The self-organization of amphiphilic molecules into unilamellar vesicles in water medium has been demonstrated. When exposed to slightly acidic pH, vesicles disorganize. This can be helpful for the cargo release after endocytosis at the early endosome stage. Dendrimer vesicles have been shown to encapsulate chemodrugs methotrexate and doxorubicin as well as to bind anti-cancer small interfering RNA Mcl-1 by means of electrostatic interactions. In summary, we have designed a novel drug delivery platform based on amphiphilic carbosilane dendrimers and proven its loading with therapeutically relevant cargo. The biological activity of the constructions obtained is now under study.

This work was supported by RFBR grant No. 18-33-20109 and by the grant of the President of

Russian Federation No. MK-2278.2019.4.

The structure of electrospun mats made of polylactide and bovine serum albumin

Bagrov D.V.1,2, Pavlova E.R. 2, Sokolova A.I. 1,2, Nikishin I.I. 2

1Lomonosov Moscow State University, Faculty of Biology, Dept. of Bioengineering. 119234, Moscow, Leninskie

gory, 1-12

2Federal research and clinical center of physical-chemical medicine of federal medical biological agency,

119435, Moscow, Malaya Pirogovskaya, 1a

Polylactide (PLA) is a biodegradable polyester used in biomedical applications, including bone screws and drug carriers. However, it has several disadvantages which limit its potential. These disadvantages include hydrophobicity, high rigidity, low degradation rate, and poor biocompatibility (relatively poor cell adhesion if compared to collagen and glass). Blending PLA with hydrophilic proteins seems a promising way to overcome these disadvantages. We hypothesized that soluble proteins, obtained from blood (e.g. albumin, fibrinogen), may be preferential to the structural ones (e.g. collagen, elastin), commonly used for this purpose. Previously we obtained electrospun mats made of a polyester, poly(hydroxybutyrate-co-hydroxyvalerate) and bovine serum albumin BSA blends and showed that these mats had unusual structural features and could release the protein gradually (Pavlova E., Bagrov D. et.al., 2017). Here we examined the properties of PLA-BSA blends and focused on the miscibility of the components. The PLA-BSA blends were prepared in a common solvent, 1,1,1,3,3,3-hexafluoropropanol (HFIP), which is a fluorinated alcohol widely used for processing polyesters. The solutions were used for electrospinning, so we obtained composite non-woven mats, which consisted of 100-1000 nm fibers. Electrospinning allowed us to mimic the structure of native extracellular matrix. Moreover, electrospinning facilitated the blending of the components, since HFIP was removed quickly, so PLA and BSA molecules were trapped in a single nanometer-sized fiber. The morphology of the electrospun mats depended on the concentration and the composition of the doping solution. When the concentration of one of the components was high enough, electrospinning yielded not only round-section fibers, but also flat ribbons. This could happen due to the buckling instability (Wang L., Pai Cl.-L. et.al., 2009). The two components (PLA and BSA) could be combined in a single fiber, as previously shown for PLA-gelatin experimental system (Bagrov D., Nikishin I. et.al., 2019). We hope that the two-component polymer-protein mats will be useful as personalized wound dressings or tissue engineering scaffolds. This work was supported by Russian Science Foundation (project №19-74-00037).

Personalized approach to the cancer patients bone defects reconstruction with porous bioactive ceramic

implants

Ales S. Buyakov1,2,a, Denis E. Kulbakin2,3,b, Svetlana P. Buyakova1,2,c, Sergey S. Kulkov1,2d

1Institute of Strength Physics and Materials Science of Siberian Branch of Russian Academy of Sciences,

Tomsk; 2Tomsk State University, Tomsk; 3Cancer Research Institute of Tomsk National Research Medical Center

of the Russian Academy of Sciences, Tomsk

a)Alesbuyakov@gmail.com, b)bkulbakin_d@mail.ru, c)buyakova@ispms.tsc.ru d)kulkov@ispms.tsc.ru

P4 medicine is one of the main directions of modern development of integrated medical care based on

personalization, prediction, preventiveness and participatory of a patient. Ideology of the P4 medicine is based

on ensuring accessibility of all modern fundamental science achievements to each patient. Personalized

approach, in this case, consists not only in determining the most effective drug tactics, but also throughout all

stages of medical treatment, including development of an endoprosthesis. Personalization is of particular

importance when providing constructive medical care to patients with damage of a bone apparatus as a result

of the malignant neoplasms, genetic diseases and injuries, when an exclusively individual approach to

osteoprosthetics tactics is required: designing the osteoimplant and minimizing the resected tissue volume.

The purpose of this work is to develop a personalized approach to the bone tissue affected areas reconstruction

meeting the conditions for ensuring democratization of medical care for patients with bone apparatus diseases

and injuries.

Osteoprosthetics of the skull visceral region requires not only reconstruction of the supporting functions, but

also restoration of shape and facial expressions, and lowering the socio-psychological barrier in front of the

patient. The skull visceral region bones have the most complex geometry of an entire human bone apparatus,

which largely determine individual characteristics of the face, as a result of which the visceral region

osteoimplants cannot be serial and belong to category of customized products.

Ceramics based on aluminum and zirconium oxides have a high tolerance to chemically aggressive environments

and do not participate in any electrochemical interactions with body tissues. The modern development of

ceramic technology indicates possibility of reproducing with an oxide ceramic a three-dimensional architectonics

of the inorganic bone matrix. Hadjicharalambous C. et al. have shown that organization of an osteo-like pore

structure gives osteoconductive properties to, bioinert in other cases, oxide ceramics.

The microstructural identity of the artificial ceramic osteosubstituting material and bone tissue can be achieved

by forming a multimodal pore structure, where the pore size can vary from tenths of nanometers to hundreds of

microns, formed by varying the structural-phase state of the initial components and molding and sintering

parameters of ceramic materials. The controlled pore volume makes it possible to create both dense

endoprostheses with high strength characteristics and highly porous ceramic matrices used as carriers of cells

that can stimulate osteogenesis and proliferation of bone tissue into the implant.

The macrostructural correspondence of the endoprosthesis to geometry of the replaced bone tissue area is

achievable through the use of modern methods of precision additive molding of medical devices. So, in 2017,

joint efforts of scientists from the Institute of Strength Physics and Materials Science, Tomsk State University and

Tomsk Institute of Oncology carried out the first operation of reconstruction the face left middle zone using a

personalized ceramic osteoimplant. Further monitoring of the patient showed the absence of a postoperative or

delayed acute inflammatory reaction, fibrosis, or a negative effect in general [2].

1. Hadjicharalambous, C., Buyakov, A., Buyakova, S., Kulkov, S., & Chatzinikolaidou, M. (2015). Porous

alumina, zirconia and alumina/zirconia for bone repair: fabrication, mechanical and in vitro biological

response. Biomedical Materials, 10(2), 025012.

2. Kulbakin, D. E., Choinzonov, E. L., Mukhamedov, M. R., Buyakov, A. S., Buyakova, S. P., & Kulkov, S.

N. (2019, April). Development of personalized approach to the reconstruction of bone tissue defects

using porous ceramic osteoimplants. In IOP Conference Series: Materials Science and Engineering

(Vol. 511, No. 1, p. 012006). IOP Publishing.

Multifunctional human serum albumin or polyethyleneimine therapeutic nucleotide conjugates as a

potential theranostic agents

Dr A.S. Chubarov

Institute of Chemical Biology & Fundamental Medicine, Novosibirsk, Russia

A choice of the drug carrier is very important for the successful design of a theranostic construct. A good carrier

should enable efficient delivery and release of actuating biomolecules, and provide their optimal distribution.

Human serum albumin (HSA) is an attractive candidate for the development of drug delivery/formulation

systems due to its enhanced uptake in tumor tissue, easily controlled surface chemistry, biodegradability,

stability and etc. Herein, we report on implementing 19F-labeled homocysteine thiolactone as a functional handle

for the synthesis of a fluorinated HSA and its site-specific coupling with a chemotherapeutic agent 5-

trifluoromethyl-2’-deoxyuridine 5’-monophosphate (pTFT) to obtain a theranostic (19F MRI and fluorescence

imaging) construct PFT-Hcy-HSA-Cy7-TFT. We have developed a novel anti-cancer conjugate with disulfide and

phosphamide bonds for on-demand delivery of pTFT in response to redox and pH dual trigger. Upon addition of

glutathione, which is relatively abundant in tumor cells, cleavage of the disulfide bond occurs, resulting pTFT

drug release. Interestingly, the pTFT release from the conjugate is greatly affected by pH and is more efficient

under mildly acidic conditions (pH 5.4).

Polyethyleneimine (PEI) has been used as a polymeric carrier for macromolecular drug preparations by a number

of research groups. Due to the negatively charged cell surface, polycationic PEI and its derivatives generally

display sufficient association and internalization rates. We report a novel strategy to engineer an acid-sensitive

anticancer theranostic agent using a vector-drug ensemble. The ensemble was synthesized by directly

conjugating the linoleic acid (LA)-modified branched PEI with a TFT. LA-PEI conjugate was modified by pTFT and

the samples containing 20-70 TFT residues per PEI molecule were obtained. The cytotoxicity of PEI-LA-pTFT

conjugates decreased with increasing nucleotide content, as examined using the MTT test. Due to the presence

of 19F, TFT-based conjugates could be detected directly in the animals by 19F MRI. In addition, the presence of

the amidophosphate group allowed their detection by in vivo 31P NMR. At the same time, the use of PEI-LA-pTFT

conjugate for chemotherapeutic drug delivery is limited due to the low release of pTFT from the carrier. To

enhance the release of the drug from the conjugate in the endosomes, PEI-LA polymer was coupled with urocanic

acid (UA), which bears imidazole ring and thus can form an acid-labile P-N bond with pTFT. The PEI-LA-UA-pTFT

conjugate containing 30 residues of UA and 40 residues of pTFT was tested against the murine Krebs-II ascites

carcinoma, grown as an ascetic tumor. The intraperitoneal injection of the conjugate resulted in prolongation of

the animals’ life and to the complete disappearance of the tumor after three injections.

Development of the Tdp 1 inhibitors as drug precursors

Dyrkheeva NS1, Apartsin EK1, Zakharenko AL1, Ilina ES1, Zakharova OD1, Luzina OA2, Ryabchikova EA1, de la Mata

FJ3, Gómez R3, Venyaminova AG1, Salakhutdinov NF2, Lavrik OI1

1Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russia

2N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, SB RAS, Novosibirsk, Russia

3Department of Organic and Inorganic Chemistry, University of Alcalá, Madrid, Spain

The ability of cancer cells to recognize the DNA damage and to initiate DNA repair is one of key mechanisms of

their resistance to chemotherapy. Therefore, the search for inhibitors of DNA repair enzymes can be used as a

strategy to potentiate the cytotoxicity of existing DNA-damaging agents. Tyrosyl-DNA phosphodiesterase 1 (Tdp1)

is a promising target for antitumor therapy based on Topoisomerase 1 (Top1) poison-mediated DNA damage.

This damage occurs in DNA via formation of Top1-DNA covalent complexes, generated by Top1 inhibitors, such

as camptothecin and its derivatives (irinotecan and topotecan) that are already used as anticancer drugs. Tdp1

plays an essential role in the removal of such complexes, which leads to a decrease of efficacy of currently

available anticancer drugs. In this regard, inhibition of Tdp1 could selectively enhance therapeutic effect of

camptothecin derivatives in the treatment of certain cancers (especially drug-resistant colon, lung and cervical

cancers). In this work, we were screening a wide range of synthetic analogs of natural compounds in order to

suppress Tdp1 enzymatic activity. We determined IC50 (half-maximal inhibitory concentration) values for the

leading compounds. Next, we checked the cytotoxicity of the leading inhibitors with IC50 in the range 10 nM - 10

mkM in the tumorous and non-tumorous cell lines by MTT assay. Non-toxic substances that do not increase the

severity of the side effects of therapy are required to sensitize tumor cells to the chemotherapeutic. Further, the

ability of the non-toxic compounds to enhance the cytotoxic effect of topotecan was determined. Some

compounds had a pronounced sensitizing effect. In the experiments on complex formation of the best Tdp1

inhibitor with amphiphilic ionic carbosilane dendrons containing fatty acids we observed a good loading capacity

into this novel family dendron based micelles. For further research on whether these compounds could be

developed as tumor cell sensitizers for Top1 inhibitors we are planning to check the cytotoxicity of these

complexes “inhibitor-dendron based micelles”.

This work was supported by RFBR grants 18-33-20109 and 19-415-540002.

Porous carriers based on aluminum oxide and polymethylsiloxane polyhydrate for controlled/modulated

drug delivery

А.А. Kotlyarova1,2*, L.N. Rachkovskaya2, T.V. Popova2, P.G. Madonov2, M.A. Korolev2, A.Yu. Letyagin2, T.G.

Tolstikova1

1 Department of Medicinal Chemistry, Novosibirsk Institute of Organic Chemistry of Siberian Branch of Russian Academy of Sciences, Lavrentjev av. 9, 630090, Novosibirsk, Russia

2 Department of Experimental Pharmacology, Research Institute of Clinical and Experimental Lymphology - a branch of the Institute of Cytology and Genetics of Siberian Branch of Russian Academy of Sciences, Acad. Timakova str., 2, 630060, Novosibirsk, Russia

* Corresponding Author. E-mail: kotlyarova.anastasiya@yanex.ru Tel. +7-913-489 20 02

Motivation and Aim: In recent years, significant efforts have been directed towards the development of porous carriers as matrices for controlled drug delivery. Due to the wide range of useful properties, porous carriers are used in pharmaceuticals for many purposes. The pharmaceutical industry has in its arsenal a wide range of different sorption materials. In our work, we used meso-, macroporous alumina with polymethylsiloxane deposited on it to obtain a prolonged form of lithium citrate - a drug used in bipolar affective disorders.

Methods and Algorithms: This study assessed the comparative pharmacokinetics of a novel prolonged release dosage form of lithium citrate in outbred CD-1 white mice – males after single intragastrically administration. In the experiment mice were divided into two groups (8-10 animals each group) which were received lithium citrate (LC) (75 mg/kg) or complex based on lithium citrate, aluminum oxide and organosilicone polymer (LAP) (1120 mg/kg) once intragastrically. These doses were calculated based on lithium containing at the ratio 5,6 mg/kg. Pharmacokinetic parameters and relative bioavailability were calculated based on lithium ions concentration in serum and brain, which was measured by inductively-coupled plasma atomic emission spectrometry (ICP-AES).

Results: According to received pharmacological data of LAP the Cmax of lithium ions in serum is lower by 4,3 times, than if administration of LC, relative bioavailability of LAP is 44.41% of standard LC. Performed research has proven that combining aluminium oxide and organosilicone polymer as supportive components with lithium citrate helps to maintaining a stable lithium ions concentration in blood and brain which is important for achieving positive lithium therapy effect.

Conclusion: Today, porous carriers play an important role in the pharmaceutical industry. Using the example of changes in the pharmacokinetic profile of lithium citrate, it was found that the presence of porous mesoporous and macroporous structure in aluminum oxide is important for ensuring stable drug delivery systems. The addition of polymethylsiloxane gives hydrophobic properties to the system and increases the sorption capacity. Therefore, in the coming years, interest in porous carriers will continue to have the best materials for drug delivery systems.

Funding: This work was performed in the course of R & D, carried out within the framework of the federal target program "Development of pharmaceutical and medical industry of the Russian Federation for the period up to 2020 and beyond." The basis for R & D is a state contract on "28" August 2015 № №14.N08.12.1041.

Interaction of hydrophobic trinuclear tungsten clusters with phospholipid bilayers: a route towards functional hybrid materials

Yuliya A. Larichevaa, Ilya S. Dovydenkob, Evgeny K. Apartsinb

a Nikolaev Institute of Inorganic Chemistry SB RAS, 3, Lavrentiev ave., Novosibirsk, 630090, Russia;

b Institute of Chemical Biology and Fundamental Medicine SB RAS, 8, Lavrentiev ave., Novosibirsk, 630090, Russia;

A large variety of trinuclear M3S4 (M = Mo, W) clusters was prepared in last 30 years, and their relevance in bioinorganic chemistry and organic catalysis has been demonstrated. Functionalization of these clusters by using electro- and photochemically active ligands such as substituted bipyridines (R2bpy) to form the [M3S4Cl3(R2bpy)3]+ complexes is very promising. The non-innocent behaviour of R2bpy and the lability of chloride ligands towards further substitution (on a wide variety of ligands including biological molecules or bio-mimetic fragments) allow us to prepare new hybrid nano-scaled constructions.

Our preliminary data shows that the incorporation of an electron-dense clusters [W3S4Cl3(R2bpy)3]+ into a lipid bilayer makes it possible to study the structure of the latter using conventional TEM. This feature can be used to study the structure of artificial and natural lipid membranes. The behavior of clusters in the hydrophobic lipid environment is determined by the volume of its ligands and cluster-to-lipid ratio. Varying these two parameters makes it possible to obtain different self-assembled associates such as cluster-doped liposomes or lipid-covered crystals. By embedding clusters with desired properties into a bilayer, we can impart these properties to the entire lipid nanoparticle to form soft functional nanomaterials.

Methods of submicron and micron drug delivery carriers investigation in vivo and in vitro

Dr Maria V. Lomova

Saratov State University, Russia.

The formation of systems to solve the issues of minimized dosing and local drug injection is an

important and promising direction in the development of biophysics and medicine. Various types of microscopy are used to characterize the surface morphology of the samples. Intravital characterization methods are some of the most popular, but include a number of restrictions, mainly regarding the resolution of the resulting images. The some number of articles shows possibilities of successful encapsulation, both water- soluble and hydrophobic substances, their local dissection, the effect on the cells, as well as the remote control of them by the magnetic field. In order to better understand the mechanism of influence on the systems of targeted drug delivery on cells, and later on animals, it is necessary to look for new approaches and criteria for determining viability. The available methods have a number of limitations, for example, the need for dye labeling, as well as the absence of simultaneous determination of several parameters in vivo. The use of solutions based on color chemical reactions or reactions with the release of gases, heat, etc. most commonly used to characterize targeted drug delivery systems. It is widely known that these protocols are most dependent on the measurement conditions and require increased accuracy of the work. The method of intravital observation of animal behavior under the influence of various kinds of factors is one of the most common methods, but its multifactorial nature greatly complicates the process of obtaining a reliable result. Our research team proposes using Brillouin spectroscopy to study the effect of targeted drug delivery systems on cells in vitro. Unlabeled technologies are the most popular and versatile ways to study living systems.

Ceria nanoparticles-decorated micro- and nanocarriers for cellular drug delivery and theranostic application

Popov A.L.1, Sukhorukov G.B.2, Ivanov V.K.3

1Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region

142290, Russia.

2School of Engineering & Materials Science, Queen Mary University of London , London E1 4NS , U.K.

3Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences, Moscow 119991, Russia.

The design of novel, effective drug delivery systems is one of the most promising ways to improve the

treatment of socially important diseases. Here we reports on an innovative approach to the production of

composite microcontainers (microcapsules) for cellular drug delivery and theranostic application. Cerium oxide

(CeO2, nanoceria) nanoparticles exhibit antioxidant, radioprotective, anti-inflammatory and other therapeutic

properties. Therapeutic activity of nanoceria is related to its unusual physical and chemical properties. First of

all, a high level of oxygen non-stoichiometry provides a remarkable redox activity in inactivating a wide spectrum

of free radicals and reactive oxygen species. Additionally, СеО2 nanoparticles are able to regenerate their redox

activity and act as biocatalysts for an unlimited number of times. Furthermore, the toxicity of nanoceria is rather

low; they have a high level of biocompatibility. These properties make cerium oxide a very promising material

for safe usage in biomedical applications. It was previously shown that nanoceria has a pH-dependent redox

activity, which may be promising in the development of new drugs for cancer therapy. Additionally, nanoceria

can be judged as a promising constituent of smart hybrid materials, enhancing the therapeutic effect of existing

drugs.

Thus, the development of a unified system for the intracellular delivery of CeO2 nanoparticles and

biologically active substances can be considered as a new strategy in the treatment of socially significant diseases,

including cancer.

Plasma polymerized films for biomolecular immobilization from theoretical modeling and experimental

study

Z.I. Popov1,2, A.M. Manakhov1, E.S. Permyakova1, M.A. Visotin3

1National University of Science and Technology “MISiS”, Moscow 119049, Russia

2Emanuel Institute of Biochemical Physics RAS, Moscow 199339, Russia

3Federal Research Center KSC SB RAS, Krasnoyarsk 660036, Russia

The deposition of epoxide groups by plasma polymerization opens new horizons for robust and quick

immobilization of biomolecules on any type of substrate. However, due to the high reactivity of this group leading

to a low functionalization efficiency. The extensive experimental and theoretical investigation of plasma

synthesis of epoxide groups from a low pressure allyl glycidyl ether (AGE) plasma were carried out. The influence

of composite parameter W/F and the working pressure on the density of epoxide groups and the layer stability

was thoroughly addressed. It was found that by increasing the working pressure it is possible to sufficiently raise

the concentration of epoxide groups. The composite parameter W/F was shown to be a crucial parameter in

affecting the density of epoxides. An optimal value of W/F of around 2.3 eV per molecule leading to the highest

density of epoxides produced in the process at 15 Pa was revealed through FT-IR and XPS findings. This value

correlates well with the ab initio calculations suggesting that the lowest bond dissociation energy belongs to the

C–O bond of the epoxide ring. Therefore, in order to increase the density of epoxides deposited by plasma

polymerization, a desirable type of precursor molecule was proposed.

The grafting of carboxyl groups enhances cell adhesion and can be used for immobilization of different

biomolecules onto plasma-treated materials. The process, however, was not well optimized due to lack of clear

understanding of the mechanisms of carboxylic group incorporation into plasma and their grafting to the polymer

surface. The deposition of COOH plasma polymers from CO2/C2H4/Ar pulsed discharge has been studied

depending on the gas mixture and duty cycle. We have demonstrated that the CO2/C2H4/Ar plasma with

adjustable thickness of COOH functionalized layer and high stability of the grafted functions in water is a better

solution for the COOH surface functionalization compared to the thoroughly analyzed CO2 plasma. The

concentration of different carbon environments and the density of COOH groups have been measured by using

chemical derivatization combined with X-ray photoelectron spectroscopy. It has been found that the CO2/C2H4/Ar

plasma mainly contains ester groups (COOC), the COOH/COOC ratio being between 0.03 and 0.08. The

mechanisms of the CO2 molecule attachment to hydrocarbon chains on the polymer surface and those located

inside the plasma were modeled using ab initio calculations.

Interpolyelectrolyte complexes as formulations for drug delivery to the brain

N. N. Porfiryeva1, R.I. Moustafine1*, V.V. Khutoryanskiy1, 2*

1Institute of Pharmacy, Kazan State Medical University, 16 Fatykh Amirkhan Street, 420126 Kazan, Russian

Federation

2Reading School of Pharmacy, University of Reading, Whiteknights, PO box 224, Reading RG66AD, United

Kingdom

The treatment of brain diseases (e.g., the neurodegenerative and psychiatric diseases, epilepsy,

oncology etc.) is a very difficult process due to the presence of blood-brain and the blood-cerebrospinal fluid

barriers, which prevent drug permeation into the brain. During the last few decades, the nasal cavity has been

studied for drug delivery directly to the brain. By using intranasal administration, it is possible to bypass the

blood–brain barrier using the nasal mucosa [1].

One of the interesting approaches is the enhancement of penetration properties of auxiliary substances,

particularly polymers, for drug delivery to the brain through the nose. In pharmaceutical technology, one of the

leaders in the field of polymers is the German concern Evonik Röhm GmbH (formerly Degussa, Röhm Pharma),

which manufactures various cationic and anionic copolymers under Eudragit® trademark. The combination of

countercharged types of (meth)acrylate copolymers allows the creation of interpolyelectrolyte complexes

(IPEC). The use of IPEC as carriers for drug delivery makes possible to obtain systems with various properties [2].

In this study, we have prepared IPEC based on a pair of copolymers Eudragit® EPO and Eudragit® L100-

55 as nanoparticles. These nanoparticles exhibited good colloidal stability and mucoadhesive properties on nasal

mucosa tissue. Thus, interpolyelectrolyte complexes based on Eudragit® EPO/L100-55 nanoparticles can be used

as a formulation potentially applied in attempts to deliver drugs to the treatment of brain diseases.

1. Hongbing Wu, Kaili Hu, Xinguo Jiang, 2008. From nose to brain: understanding transport capacity and

transport rate of drugs, Expert Opinion on Drug Delivery, 5:10, 1159-1168.

2. Mustafin (Moustafine), R.I., 2011. Interpolymer combinations of chemically complementary grades

of Eudragit® copolymers: a new direction in the design of peroral solid dosage forms of drug delivery systems

with controlled release, Pharmaceutical Chemistry Journal, 45, 285–295.

Luminescent silica microparticles as new detectable agents for protein transduction

Tatiana N. Pozmogova1,2, Yuri A. Vorotnikov3, Anastasiya O. Solovieva1, Svetlana M. Miroshnichenko1, Michael A.

Shestopalov3, Olga A. Efremova2

1Scientific Institute of Clinical and Experimental Lymphology – branch of ICG SB RAS, 2 Timakova str., 630060

Novosibirsk, Russian Federation

2Novosibirsk State University, 2 Pirogova str., 630090 Novosibirsk, Russian Federation

3Nikolaev Institute of Inorganic Chemistry SB RAS, 3 Acad. Lavrentiev Ave., 630090 Novosibirsk, Russian

Federation

Keywords: microparticles, protein delivery, imaging agents, metal cluster complexes

Silica is an excellent matrix for a wide range of biomedical applications, due to its high inertness, biocompatibility

and versatility of the forms and shapes that can be achieved. For example, silica nanoparticles were successfully

used for the cellular delivery of nucleic acids, proteins, drugs against various types of diseases, imaging agents,

etc. Many of these applications require, however, multi-step modification of silica particles by luminescent tags

to allow their monitoring using conventional imaging techniques such as confocal microscopy or flow cytometry.

In this work we present (Bu4N)2[Mo6I8(NO3)6] as an excellent precursor for one-pot synthesis of highly

photoluminescent and chemically stable silica microparticles (500 nm). We demonstrate that this particles are

taken well by the larynx carcinoma (Hep-2) cells and have low dark toxicity, which is predominantly determined

by silica itself and not by the molybdenum cluster. The photoinduced cytotoxicity is also considerably low due to

the low surface area. Notably, the particles can be further easily modified to allow attachment of a biological

cargo. Specifically, the surface of silica microparticles were functionalised with epoxy groups to allow successful

grafting and transduction of a model protein – Green Fluorescent Protein. It was determined that protein keeps

its biological properties after being attached to the surface of microparticle. Cellular uptake and localisation of

microparticles with and without cargo was studied and compared. In summary, molybdenum cluster modified

silica offers high potential for protein transduction or other types of traceable cellular delivery as well as in

multimodal theranostic applications.

This work is supported by the Russian Foundation for Basic Research grant №18-315-00235

Quantitative and structural characteristics of mitochondrial DNA in varicose veins of lower extremities

Smetanina M.A.1,2, Shadrina A.S.1,2,3, Sevost'ianova K.S.1,2, Shirshova A.N.1, Oscorbin I.P.1, Oskina N.A.1,

Zolotukhin I.A.4, Filipenko M.L.1,2

1Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia; 2Novosibirsk State University,

Novosibirsk, Russia; 3Institute of Cytology and Genetics, Novosibirsk, Russia; 4Novosibirsk Pirogov Russian

National Research Medical University, Moscow, Russia

Background/Objective: Varicose veins of lower extremities (VVs) are a highly prevalent venous pathology. Despite of this, our understanding of the molecular mechanisms underlying the pathogenesis of this condition is yet to be clarified. Venous wall composition and morphology of its constituents is substantially changed during vein wall remodeling. Smooth muscle cells of the middle layer that are responsible for venous tone change their contractile function to a secretory one, which may correspond to a decrease in mitochondria in the cells since there is no much need to get energy from them. The aim of our study was to provide quantitative (in terms of mtDNA/nuclear DNA) and structural (in terms of common deletions in the MT-ND4 gene region) characteristics of mitochondrial DNA in VVs by comparing mitochondrial genome parameters in varicose and non-varicose vein tissue samples.

Methods: The study sample consisted of 60 individuals with VVs of C2-C4 class according to CEAP. Total DNA was extracted from paired samples of varicose and non-varicose segments of great saphenous veins left after venous surgery (120 samples, each pair of samples from the same patient). Relative mtDNA level and the proportion of mtDNA without common deletions were determined by a multiplex real-time quantitative polymerase chain reaction assay. Differences between pairs of samples were analyzed by the Wilcoxon signed-rank test.

Results: We observed that total mtDNA level was lower in VVs than in non-varicose samples (N = 60; P = 0.004; median non-VVs/VVs ratio = 1.19). Differences in the proportion of no-deletion mtDNA were not statistically significant (P = 0.08). When the study sample was stratified by age, Bonferroni-corrected threshold of statistical significance was reached only in women (N = 43) and only for the differences in mtDNA level (P = 0.002; median non-VVs/VVs ratio = 1.20).

Conclusion: For the first time, our results provide evidence that changes in the level of mtDNA can be involved in VVs pathogenesis. We suppose that decreased mtDNA level in varicose veins compared to non-varicose vein segments can be related to phenotype switching of smooth muscle cells and loss of their contractile capacity leading to a loss of venous tone. Since this study was not designed to draw causal inferences, it is not clear now whether decline in mtDNA level results from a pathological remodeling of the vein wall or it is one of the factors underlying VVs development. Further studies would be beneficial to elucidate the role of mitochondrial function and genome parameters in VVs pathogenesis.

The work was supported by the Russian Science Foundation (Project 17-75-20223 “Investigation of the mechanisms of vein wall remodeling in varicose veins”).

Modification of PCL nanofibers for a wide range of tasks in regenerative medicine

A.O. Solovieva1*, S.M. Miroshnichenko1,2, A.M. Manakhov1

1 RICEL– Branch of the ICG SB RAS, Novosibirsk, Russia

2FRC of FTM - Institute of Biochemistry, Novosibirsk, Russia

Polycaprolacton (PCL) is a synthetic material suitable for biomaterials development due to its improved reproducibility between batches and mechanical properties, which are more easily tuned. It is also often less expensive to produce on industrial scale. ECM-like structure of PCL nanofibers obtained by electrospinning methods is an important parameter for the purpose of tissue engineering. On the other hand, nanofibers are superhydrophobic and biologically inert which considerably complicates the cell adhesion and affects their further fate negatively. There are many approaches to enhance the biocompatibility of nanomaterials: cospinning with natural and synthetic polymers, soaking of the matrices in the extracellular matrix proteins etc. One of the most powerful and promising strategies for generating advanced surface of PCL nanofibers is the deposition of COOH functional groups by cold plasma polymerization.

We study the deposition of plasma polymer films containing functional COOH groups followed by immobilization of proteins. Using the modified surface of PCL fibers with COOH functional groups allows physiological binding of proteins without loss of their functional activity. It is possible to achieve a stronger covalent bond that allows to dose and prolong the action of protein molecules when using a linker (EDC).

It is shown that the modification of the PCL nanofibers with the COOH plasma polymers and the subsequent binding of protein molecules (platelet rich plasma - PRP) is a rather simple and technologically achievable procedure, which allows the adhesion, early spreading, and growth of human fibroblasts as well as mesenchymal stem cells boost. The covalently bound components of PRP considerably reduce the MSC and fibroblast apoptosis and increase the cell proliferation in comparison to the unmodified PCL nanofibers or the PCL nanofibers with non-covalent bonding of PRP. Soaking of PCL ref scaffolds in the PRP was improved by cell proliferation and viability, but still short in comparison with ionic and covalent PRP bonding scaffolds.

The reported research findings reveal the potential of plasma modification with COOH groups PCL matrices for application in tissue engineering. The subsequent covalent protein binding with surface expands this potential even further.

This work was funded by the Russian Science Foundation (grant No. 18-75-10057).

Biomaterials for Drug Delivery and Development

Veniamin Khazanov

Innovative Pharmacology Research ltd., Tomsk, Russia

Biomaterials are compounds developed for interaction with the human organism. They may have different chemical nature (organic and nonorganic) and origin (natural and synthetic), and also may be biologically active or inert. Their influence on a drug’s properties isn’t limited to the delivery of active compound. They also affect drug production technology (solubility, flowability, compressibility, bulk density, gliding, uniform dosage, stability, form keeping), consumer qualities (shape, color, taste, smell, convenience of use, package) and main pharmacological properties (efficacy, safety and pharmacokinetics). Drug development is based on optimal selection of biomaterials and technologies for obtaining a drug with the desired safety and efficacy in a convenient dosage form. The problems of wrong choice of biomaterials can become apparent in transferring laboratory technology to industrial production, in preclinical and, most dangerously, in clinical trials. The selection of the best biomaterial for a drug is more art than science: existing in silico methods aren’t optimal enough for this process. A properly formed pharmaceutical development plan, combined with preclinical and clinical trials, allows to implement an innovative drug project in 10-12 years. Poor choice of biomaterial can send a project back from clinical stage to preclinical and destroy the manufacturer plan of investment – to get a profit in the patent protection period. This can lead to a complete closure of the project due to insufficient profitability.

Shining a Light on Stem Cell Differentiation: Photo-patterned Supramolecular Hydrogels for Tissue Engineering

Phillip R. A. Chivers,a,b Simon J. Webb,b Paul G. Genever,c and David K. Smith*a

a Department of Chemistry, University of York, United Kingdom. b Manchester Institute of Biotechnology, University of Manchester, United Kingdom. c Department of Biology, University of York, United Kingdom E-

mail: phillip.chivers@manchester.ac.uk

The high demand for transplant tissue is a major health concern worldwide. A shortage of donors combined with poor long-term outcomes post-transplant has seen tissue engineering emerge as an alternative therapy. Using a patient’s own stem cells to regenerate lost or damaged tissue could reduce pressure on transplant waiting lists and avoid issues of organ rejection. However, for laboratory research to be translated into clinical applications, the development of biomaterials which stimulate the formation of specific tissues is essential.1

In recent years, hydrogels have become leading candidates as scaffolds for controlled cell growth. Despite this, there is a dearth of literature exploring the applications of low-molecular-weight gels (LMWG) in this field.2 These materials, which self-assemble through non-covalent interactions, are often dynamic – able to modulate their structure and properties in response to their external environment. Such responsive materials have great potential for complex control of stem cell fate through the delivery of spatiotemporally defined cues.3

We report the use of LMWG-containing materials for the spatial control of mesenchymal stem cell (MSC) behavior. By combining DBS-CONHNH2 (a LMWG) with a photo-curable polymer gel (PEGDM), we were able to modify the stiffness of the gels with spatial control by exposing specific regions to UV irradiation.4 MSCs grown on stiffer gels were significantly more likely to differentiate into bone cells than those cultured on soft gels. Furthermore, MSCs at each side of an interface between soft and stiff gel showed significantly different behavior, illustrating the potential of these patterned materials to direct stem cell growth. Studies into the controlled release and diffusion of model compounds and biomolecules indicate that these heterogeneously structured materials could be used for spatially-defined presentation of other stem cell directing factors in the future.

Temporal changes in gel stiffness can be used to impart a further level of control over MSC behavior. We are currently developing a family of hydrogels which modulate their mechanical properties through a reversible photoreaction. Using these materials, we aim to understand and exploit the influence of stem cell mechanical memory to develop next-generation biomaterials.

References

1 Webber, M. J.; Appel, E. A.; Meijer, E. W.; Langer, R. Nat. Mater., 2016, 15, 13-26

2 Christoff-Tempesta, T.; Lew, A. J.; Ortony, J. H. Gels, 2018, 4, 40

3 Chivers, P. R. A; Smith, D. K. Nat. Rev. Mater. 2019, 4, 463-478

4 Chivers, P. R. A; Smith, D. K. Chem. Sci. 2017, 8, 7218-7227

Figure 1. Spatially-resolved hydrogels can spatially control MSC growth and small molecule diffusion

Middle (shielded from UV) = Soft gel

Outer edge (exposed to UV) = Stiff gelStiff gel

Soft gel