Technology

Introduction

Gene therapy is the delivery of therapeutic nucleic acids (genes) into cells of a patient to treat a disease. By adding a corrected copy of a defective gene, gene therapy can help diseased tissues and organs to work properly. Gene therapy is fundamentally different from traditional drug-based approaches, which may treat symptoms but not the underlying genetic problem. Gene therapy has a tremendous potential for treatment of all kinds of diseases since more than 8,000 human diseases are due to gene errors. However, the progress of gene therapy is strongly hampered by the lack of safe and efficient carriers that can deliver the therapeutic genes into the cytoplasm of diseased cells. Viral gene delivery systems are currently investigated in various clinical trials and even in use in a number of applications, but viral delivery systems have some serious drawbacks as these are difficult to produce, have only limited loading capacity, and raise serious safety concerns due to potential adverse immune responses. For a wide area of clinical applications there is an urgent need for safe and efficient non-viral gene delivery systems that efficiently bring therapeutic genes into target cells.


20Med Nanogels

20Med Nanogels are well-processable, nontoxic and biodegradable nanoparticles that are stable in solution or can be stored as powder formulation. 20Med Nanogels have an open, flexible cage-like structure in its unloaded form. Functional groups inside the core of the nanogels enable easy and efficient loading of (oligo)nucleotides by simply mixing of the components in aqueous solution. After loading, the cage collapses and protects the therapeutic payload from degradation in the extracellular environment. The surface of the nanogels can be easily provided with a stealth coating and targeting groups for cell specific uptake. Chemical functionalities present in the 20Med-nanogels promote escape of the nanogels from the endosomes to avoid the degradation pathway. Once arrived in the cytoplasm, the polymeric network of the nanogels is subject to rapid biodegradation with the result that the therapeutic (oligo)nucleotides are efficiently released into the cytosol, giving high transfection and high gene silencing activity.

20Med Nanogels can be loaded with 100% efficiency by simply mixing of (oligo)nucleotides with nanogels in buffer solution
Figure 1. 20Med Nanogels can be loaded with 100% efficiency by simply mixing of (oligo)nucleotides with nanogels in buffer solution. Loaded nanogels can be post-modified with polymeric surface coating and targeting moieties to give the nanogels stealth properties and cell specificity. After cellular uptake, different chemical functionalities in the nanogels promote endosomal escape of the nanogels by which the nanogels avoid the natural digestive pathway and arrive in the cytosol. There the polymeric network of the nanogel is broken down and the therapeutic payload is released in the cytosol to display its functional activity.


20Med Nanogels have already proven to be highly efficient in gene transfection in a broad range of cells. Due to their special chemical construction, 20Med Nanogels are very flexible in tuning their chemical functionalization and molecular architecture. This makes the nanogel platform also suitable for developing tailor-made carriers for targeted delivery of therapeutic peptides, proteins, and small molecules.

Key characteristics of 20Med Nanogels

Figure 2. Illustration of the great tunability of the 20Med Nanogels.


Key characteristics of 20Med Nanogels:
  • can be easily prepared with controlled size in the range 50-200+ nm without the need for excess of polymer or the presence of therapeutics in solution.
  • can be post-loaded with (oligo)nucleotides by simply mixing nanogels and (oligo) nucleotides in aqueous solution.
  • are stable in solution and can be lyophilized and stored as powder formulation without loss of integrity and activity.
  • are non-toxic, non-immunogenic, and biodegradable.
  • protect therapeutic payload from degradation by nucleases.
  • show efficient endosomal escape and efficient release of therapeutic payload into the cytoplasm, resulting in high intracellular activity.
  • can be coated with stealth and targeting moieties and targeting groups can be attached.
  • allow modification of the core of the nanogels with complementary binding sites of other drugs (peptides, proteins, small molecules) as well as for drug combinations.


20Med Nanogels can be loaded with high amounts of siRNA

20Med Nanogels can be produced in different sizes, ranging from 50 nm to larger than 200 nm. The nanogels have a high nucleotide loading capacity. For example, a single nanogel of 120 nm can be loaded with 100% loading efficiency with 108 siRNA molecules per nanogel particle. No leakage of siRNA from the nanogel occurs in the extracellular environment, whereas siRNA is rapidly released in the cytosolic plasma.

20Med Nanogels can be stored without loss of activity

Both loaded and unloaded nanogels are stable nanoparticles that retain full activity in solution at room temperature, after freeze-thawing cycles and after storage as freeze dried powder. No aggregation of the nanogel particles occurs. As is shown in Figure 3 all three siRNA loaded nanogel formulations showed equal efficiency in the knock down of the target luciferase gene.

Anti-LUC siRNA activity to H1299 cells by siRNA/ 20Med Nanogels as freshly prepared formulation, freeze-thawed formulation and from freeze-dried siRNA/ 20Med Nanogel powder dissolved in buffer solution. The control is a freshly prepared
					siRNA/ 20Med Nanogel formulation with a non-coded siRNA.

Figure 3. Anti-LUC siRNA activity to H1299 cells by siRNA/ 20Med Nanogels as freshly prepared formulation, freeze-thawed formulation and from freeze-dried siRNA/ 20Med Nanogel powder dissolved in buffer solution. The control is a freshly prepared siRNA/ 20Med Nanogel formulation with a non-coded siRNA.


20Med Nanogels are non-toxic and non-immunogenic

An important requirement for gene delivery vehicles is the absence of significant toxicity. MTT and LDH assays show that 20Med Nanogels are nontoxic for cells in the concentration range usually used for oligonucleotide delivery as they are rapidly degraded once arrived in the cell plasma. In preliminary in vivo experiments no toxicity and immunogenicity is observed.

20Med Nanogels are highly efficient in intracellular delivery

20Med Nanogels loaded with siRNA are highly efficient in gene silencing. They display high efficiency in delivery of nucleic acids into the cell, due to a unique combination of properties, such as high endosomal buffering capacity, which provides high endosomal escape to avoid inactivation in the lysosomal pathway. Fast biodegradation of the nanogel polymeric network delivers siRNA in its free, functional active, form in the cytoplasm. 20Med Nanogels incorporating anti-GFP-siRNA show very efficient knock down of the production of GFP in various cell lines, both in serum free and in serum containing medium (Figure 4).

Effect of different formulations of anti-GFP siRNA in 20Med Nanogels on gene silencing of GFP expressing RAW cells.

Figure 4. Effect of different formulations of anti-GFP siRNA in 20Med Nanogels on gene silencing of GFP expressing RAW cells.
Left: Quantification of nanogel formulation showing that sub-picomolar concentrations per well ( <10-12M) give already strong gene silencing. Right: Fluorescent microscopy images showing efficient silencing of GFP production in RAW cells.


GFP expression and percentage of GFP-positive cells in Cos-7, RAW264.7, and U87 cells after transfection with 20Med Nanogels loaded with eGFP messenger RNA.

Figure 5. GFP expression and percentage of GFP-positive cells in Cos-7, RAW264.7, and U87 cells after transfection with 20Med Nanogels loaded with eGFP messenger RNA.