PECTIN-BASED INJECTABLE DELIVERY SYSTEMS FOR REGENERATIVE MEDICINE

Transcription

1 PECTIN-BASED INJECTABLE DELIVERY SYSTEMS FOR REGENERATIVE MEDICINE Maria Cristina Tanzi INSTM local unit Politecnico di Milano. Dept. Of Chemistry, Materials and Chemical Engineering «G. Natta», Milano, Italy

2 OUTLINE 1. What is pectin? 2. Biomedical applications of pectin gels 3. Injectable gels by internal gelation mechanism 4. Model L929 and hadscs immobilization 5. Injectable antimicrobial gels

3 Pectin: an overview Natural sources Primary cell wall Chemical structure Poly-galacturonic acid 2

4 Pectins: esterification degree 4 Degree of esterification: esterified/non-esterified galacturonic acids HM Pectins LM Pectins We use a low methoxyl (LM) pectin from citrus fruits (CU701) (Herbstreit & Fox Neuenbuerg, Germany)

5 LM pectins: Gel formation LM pectins ph ( ) by positive divalent ions egg box structure Branched regions COOH COOH COOCH 3 COOCH 3 COOH rhamnogalacturonan-i rhamnogalacturonan-ii 3

6 Pectin properties for biomedical applications Simple and cytocompatible gelling mechanism Flexibility of fabrication in different shapes Versatility Physiological-like environment Sterilizability INJECTABLE GELS MICROSPHERES 4

7 Past work: Injectable microspheres Gels are formed by fast gelation (CaCl2) Microspheres Delivery systems (drugs, genes, cells, proteins, growth factors, other biomolecules)

8 Past work: Microspheres preparation Pectin gelling Pectin + cell suspension Egg-box structure a) Extrusion with a coaxial air flow system A) 250 um b) Electrostatic system FAST gelation = External gelation

9 This work: Injectable pectin gels 1

10 Injectable hydrogels homogeneity of immobilization minimal invasivity adaptation to the defect shape viscoelastic materials protect cells from mechanical damage during extrusion 11

11 Cell immobilization no polarity induced by cell-matrix interactions as in the 2D condition Cell immobilization in a 3D structure spatial distribution of cells into the gel cell segregation due to the low viscosity slow gelling kinetic rapid gelling kinetic inhomogeneous distribution of the cells 13

12 Aim of the study Design of injectable pectin hydrogels with controlled gelling kinetics, to: tailor different surgical procedures and anatomical sites supply cell viability for soft tissue regeneration obtain hydrogels with easy manufacturing techniques and at low production costs Development of pectin gels by internal gelation with: different formulations adequate injectability tunable mechanical properties shelf-life stability biocompatibility and cytocompatibility 14

13 Preparation of the hydrogels Internal gelation approach Raising the acidic ph of pectin solutions is needed to obtain the best compromise for the internal gelation mechanism, which requires acidic environments, and for biocompatibility of the produced gels, that implies a ph close to the physiological one Reversibility of the gelling process by dissolving gel with a saline solution (NaCl) H. Moreira, F. Munarin, R. Gentilini, L. Visai, M.C. Tanzi, P. Petrini:, Carbohydrate Polymers, 2014, 103,

14 Preparation of the hydrogels Internal gelation approach Reversibility of the gelling process by dissolving gel with a saline solution (NaCl) H. Moreira, F. Munarin, R. Gentilini, L. Visai, M.C. Tanzi, P. Petrini:, Carbohydrate Polymers, 2014, 103,

15 Hydrogels characterization Rheological tests Swelling study ph measurement Cytotoxicity tests Characterization to evaluate their injectability and stability 16

16 Hydrogels rheological characterization GEL POINT: intersection of storage (G ) and loss moduli (G ) In the case of the gel obtained with 23 mm NaHCO 3 and12.5 M CaCO 3 (PNa23Ca12.5) the gel point is reached in 17 min H. Moreira, F. Munarin, R. Gentilini, L. Visai, M.C. Tanzi, P. Petrini:, Carbohydrate Polymers, 2014, 103,

17 Gelling times as a function of NaHCO 3 and CaCO 3 Note: [CaCO3] = 12.5, 25 and 50 mm correspond to R = 1, 2 and 4 respectively, where R=[Ca ++] /2[COO - ]

18 Hydrogels rheological characterization ph 17

19 Rheological characterization SOFTER GELS ph ADEQUATE FOR SOFT TISSUE REGENERATION EASIER CONFORMING TO THE ANATOMICAL SITE STIFFER GELS INJECTABLE FORMULATIONS FOR THE REPAIR OF HARD TISSUES 3D STRUCTURAL NETWORK FOR THE CELLS 18

20 soft tissue regeneration SOFTER GELS EASIER CONFORMING TO THE ANATOMICAL SITE Double effect Passive as a filler of the defect Active as a cell carrier 18

21 Adipose tissue regeneration Esthetic/cosmetic: small lipoatrophies Large lipoathophies WHEN Pectin + Adipose derived stem cells Gel formation Soft tissue regeneration Gel degradation in vivo and cell release 19

22 Pectin-based hydrogels Production and characterization Optimization of the gel formulation and of the process of cell immobilization 21

23 The approach: experimental steps A) dual syringe system to mix the pectin solution added with cells and the crosslinker CaCO 3 ; (23mM NaHCO 3, 50 mm CaCO 3 ) B) prior to gel, the mixing solution is poured in each well and C) after reaching the gel point, medium culture is added, until the cells are entrapped in the gel network(d).

24 The approach: bioactive additives Pectin solution CaCO 3 suspension simple, non-immunogenic and non-patient specific molecules glucose glutamine 20

25 Hydrogels formulation Study of dose dependence of glucose and glutamine Nomenclature NaHCO 3 (mm) Glucose (mm) Glutamine (mm) Pe0Ca PeCa PeCaglc PeCagln PeCaglc / PeCagln / PeCaglc /2 +gln / PeCaglc + gln PeCa 2glc + 2gln

26 Rheological characterization Storage modulus (Pa) a) Frequency (Hz) A) Frequency (Hz) Complex viscosity (Pa s) B) Tan (NO Cells) Frequency (Hz) Pe0Ca PeCa PeCa glc PeCa gln PeCa glc /2 +gln /2 PeCa glc+gln PeCa 2glc+2gln Storage modulus (Pa) (+ L929 Cells as a model line) Complex viscosity (Pa s) a) Frequency (Hz) Frequency (Hz) PeCa PeCa with cells PeCa glc+gln PeCa glc+gln with cells Storage modulus and complex viscosity (inset a) of the hydrogel samples with and without immobilized L929 cells 23

27 Live dead assay with L929 Pe0Ca 100 µm PeCa 100 µm Low cell viability High cell viability and homogeneous distribution Live/Dead assay: Calcein / Propidium Iodide, confocal scanning laser microscopy

28 In vitro characterization (1): L929 murine cells Cell viability on the different gel formulations up to 24 hours Trypan blue assay *Cell viability = (live cells / tot cell number)*100 CLSM analysis 24

29 In vitro characterization (2): ): L929 murine cells Cell viability with increase of glutamine and glucose concentrations up to 24 hours of immobilization Trypan blue assay CLSM analysis 25

30 Pectin-based hydrogels Production and characterization Optimization of the gel formulation and of the process of cell immobilization Characterization using different needles of extrusion Injectability and cell viability 26

31 Rheological characterization The extruded gel from different needles maintains a frequency independent and a shear thinning behavior no needle = 1.47 mm 20 G = 0,7 mm 25 G = 0,34 mm 30G = 0,18 mm 27

32 Injectability characterization: texture analysis 50 N is the maximum force tolerable to minimize patient discomfort Increase of the injection force associated to the reduction of the needle diameter Texture analyzer (injection rate 100mm/min until a distance of 30 mm) 28

33 In vitro characterization: adipose derived stem cell from human patient (Trypan blue) * *Cell viability = (live cells / tot cell number)*100 no needle 20 G 25 G Live/Dead assay: Calcein / Propidium Iodide 29

34 ADSC morphology and cytoskeletal structure (7 days of incubation) MSC on coverslip MSC on PeCa gels 100 µm 100 µm tubulin: green actin: red nuclei: blu (good preservation of the morphology) 30

35 Human ADSCs maintenance of stemness (7 days of incubation) MSC on coverslip 100 µm MSC on PeCa gels 100 µm CD90: green CD44: red CD146 green STRO1: red (stemness markers) Nuclei: blu 31

36 Antimicrobial hydrogels Injectable antimicrobial hydrogels for the treatment of implant-related infections 32

37 Introduction Surgical Site Infections (SSI) account for 20% of all health care-associated infections in U.S. hospitals 1 estimated 8,205 annual deaths caused by SSIs 1 780,000 SSIs occur each year 2 35,000 SSIs develop annually after orthopedic surgery 2 up to 20,000 knee and hip replacement patients contract an SSI 3 Traditional approaches are limited to systemic antibiotic treatment that requires higher doses of antibiotics for long periods of time to try to cure the infection at the implant site. Moreover, high systemic levels of antibiotics come with significant side effects, including life-threatening allergies and organ failure 1. Klevens RM, Edwards JR, Richards CL Jr, et al., World Health Organization. WHO Guidelines for Safe Surgery, Greene LR

38 Antimicrobial Peptides (AMPs) Naturally derived molecules part of the innate immune system of all multicellular organisms Potential to be an alternative to antibiotics, exhibiting broad-spectrum activity against bacteria, viruses, and fungi Indolicidin (13 aa) Chrysophsin-1 ( -helical cationic peptide Maria Cristina Tanzi 9th A.It.U.N. Annual Meeting, May 25-27,

39 Aim of the study Design of injectable antimicrobial hydrogels in implant-related infections + Pectin Antimicrobial Peptides (AMP) Grafting NHS/EDC carbodiimide chemistry -- Bacterial action of the AMP released from pectin hydrogel Injection of antimicrobial hydrogel in the infected tissue 35

40 Hydrogel formulations Maria Cristina Tanzi 9th A.It.U.N. Annual Meeting, May 25-27,

41 FT-IR spectroscopy increase of the band at 1610 cm -1 (amide II) increase of the band at 1420 cm -1 (amide II) vibrations of the N H bending and the C N stretching the effects of primary and secondary amides absorption. Wavelenght (nm) N-H stretching: peptide bond CHY 10 INDO 10 LM pectin Wavelenght (nm)

42 Bacteria reduction assay INDO pectin with 1 µm and 10 µm of indolicidin CHY pectin with 0.1 µm and 1 µm of chrysopsin Bacteria reduction (%)= (#CFU crl -#CFU sample )/(#CFU crl )

43 From lab to product. ability to heal the site of infection in a faster way than traditional approach injection directly in the infected tissue reducing the total hospital stay and the medical cost 39

44 Acknowledgments Roberta Gentilini, PhD in Biomedical Engineering 2014 PhD Thesis: Pectin hydrogels for regenerative medicine: injectable systems, cell delivery and antimicrobial formulations Biomaterials Lab, Politecnico di Milano Molecular Medicine Department, Pavia University Life Sciences and Bioengineering Center, Worcester Polytechnic Institute, USA (Prof. Terry Camesano)

45 Acknowledgments - Helena R. Moreira - Mário A. Barbosa - Pedro L. Granja