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Current sustained delivery strategies for the design of local neurotrophic factors in treatment of neurological disorders
a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 2 6 9 e2 7 7
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Review
Current sustained delivery strategies for the design of local neurotrophic factors in treatment of neurological disorders Hongzhuo Liu a, Yanyan Zhou a, Shichao Chen b, Meng Bu c, Jiayu Xin c, Sanming Li a,* a
School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, China School of Life Science & Biopharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China c Harbin Medical University, Harbin 150081, China b
article info
abstract
Article history:
Although therapeutic potential of neurotrophic factors (NTFs) has been well recognized for
Received 19 August 2013
over two decades, attempts to translate that potential to the clinic have been disap-
Received in revised form
pointing, largely due to significant obstacles in delivery, including inadequate protein dose/
8 October 2013
kinetics released at target sites. Considerable efforts have been made to improve the
Accepted 9 October 2013
therapeutic performance of NTFs. This articles reviews recent developments in localized delivery systems of NTFs for the neurological disorders treatments with a main focus on sustained delivery strategies. Different non-covalent binding approaches have been
Keywords:
employed to immobilize proteins in hydrogels, microspheres, electrospun nanofibers, and
Neurotrophic factors
their combined systems, which serve as depots for sustained local release of NTFs. The
Neurological disorders
challenges associated with current NTFs delivery systems and how these systems can be
Drug delivery
applied to neurological diseases and disorders have been discussed in the review. In
Sustained delivery
conclusion, optimal delivery systems for NTFs will be needed for reliable and meaningful
Release kinetics
clinical benefits; ideally, delivering a time and dose-controlled release of bioactive multiNTFs at different individual optimal kinetics to achieve multi-functions in target tissues is significant preferred. ª 2013 Shenyang Pharmaceutical University. Production and hosting by Elsevier B.V. All rights reserved.
1.
Introduction
Neurotrophic factors are polypeptides primarily known to regulate the survival and differentiation of nerve cells during the development of the peripheral and central nervous
systems. Over the past two decades, scientists have had great expectations for NTFs in the treatment of neurological disorders, including the most common central neurodegenerative diseases, Alzheimer’s (AD) and Parkinson’s (PD) [1]. This approach may also prove effective for the peripheral nerve
* Corresponding author. Tel.: þ86 24 23986258; fax: þ86 24 23986293. E-mail address: [email protected] (S. Li). Peer review under responsibility of Shenyang Pharmaceutical University
Production and hosting by Elsevier 1818-0876/$ e see front matter ª 2013 Shenyang Pharmaceutical University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ajps.2013.10.003
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injury, which is a serious issue affecting 2.8% of trauma patients and many of whom will be subjected to life-long disability [2]. Moreover, NTFs also play an important role in the development of potential neuroprotective glaucoma and hearing disorders treatments [3e5]. Despite their obvious attractiveness as therapeutic agents, they have some serious restrictions in clinical use. The most limiting of these is their short biological half-life and vulnerability to structural disruption or modification, leading to loss of bioactivity. Therefore, protein drug delivery systems are needed not only to improve the biological utilization by sustained release of bioactive NTFs to target site at sufficient concentration, but also to protect their bioactivity from degradation by direct exposure to harsh environments. There has been a growing interest to develop new strategies for effective delivery of NTFs. This article reviews some widely investigated NTFs and their delivery systems, summarizes the difficulties in their neurodegenerative diseases therapy and the most pertinent evaluation system for testing delivery systems, and finally provides perspectives for advanced nerve repair strategies that may hold promise for enhancing therapeutic efficacy.
2.
Neurotrophic factor
Nerve growth factor (NGF), discovered by R. Levi Montalcini almost 60 years ago, is regarded as the first discovered and as an important member of neurotrophin family [6]. Later, three NGF homologous growth factors were identified: the brainderived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4/5. Each has its distinct and/or overlapping activities within the developing peripheral and central nervous systems and are collectively indicated as neurotrophins [7]. Widely studied NTFs also include the glial cell line-derived neurotrophic factor (GDNF, belonging to the transforming growth factor-b superfamily), the glial growth factor (GGF) (a member of the neuregulin family), neuropoietic cytokines [such as ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF)] and other cytokines [2]. Since important NTFs in adulthood as well as development, reducing the level of one or more of these proteins may be responsible for the neurological disorders. Each of the factors has been employed in the neurorestorative therapy in some form or another in clinical trials. To brief illustrate, neurotrophin family and GDNF are addressed when selecting or designing a delivery system in this review. Although GDNF shows only limited amino-acid sequence homology with the member of neurotrophins family, it has somewhat conformational similarity as they all have the characteristic cystine knot structure motif with the formation of three disulphide bonds. The above proteins differ in their spectrum of action when applied as a single factor. NGF is essential for the development and phenotypic maintenance of neurons in the peripheral nervous system (PNS) and for the functional integrity of cholinergic neurons in the central nervous system (CNS) [1]. It has been considered as a very powerful and selective growth factor for sympathetic and sensory neurons, especially primarily survival and axonal outgrowth of sensory neurons [8]. Meanwhile BDNF supports the survival and maintenance of sensory neurons, retinal
ganglia, certain cholinergic neurons, spinal motor neurons and some dopaminergic neurons [1]. NT-3 helps to support the survival and differentiation of existing neurons, and also encourages the growth and differentiation of new neurons and new neurons and synapses [1]. GDNF shows pronounced effects on the survival of midbrain dopaminergic neurons. Even more interesting, it is identified to be key factor for motor axonal regeneration for its ability to promoting axonal elongation [9,10]. Neurotrophin family binds to two different receptors: high affinity binding via tropomyosin receptor kinase (Trk)-A, B or C and low affinity nerve growth factor receptor (p75 neurotrophin receptor, P75NTR). NGF binds TrkA; BDNF, NT-4/5 binds TrkB; NT-3 binds TrkC. Finally, the binding of GDNF by its homodimeric receptor tyrosine kinase complex leads to activation of the TrkA pathway, which in the case of GDNF is highly complex and interdependent [1].
3. Current delivery strategies for neurotrophic factors Though applied to a wide spectrum of neurological diseases, the clinical utilization of NTFs, , remains limitations by important adverse events induced by exposing non-targeted tissues [11] coupled with the mild side effects like those derived from the effects of NGF on pain system [12]. These effects are especially when systemically administration. Moreover, NTFs undergo very short biological half-life in circulation and rapid degradation in vivo, and also present poor permeability across the biological barriers [11]. Therefore, to an efficacious clinical outcome, localized delivery for NTFs is preferred. Nevertheless, the clinical outcome remained, however generally unsatisfactory, and was probably due to inadequate NTFs doses and/or release kinetics at the optimal location, undesired initial burst release, and the use of single NTFs rather than multiple factors as occurs naturally [2]. There have been many attempts to deliver various NTFs to target sites by using diverse types of synthetic and natural materials in order to modulate the release kinetics. Generally, the proteins were designed to be immobilized in the systems through non-covalent or covalent means.
3.1.
Non-covalent neurotrophic factors binding
Non-covalent binding includes physical entrapment, adsorption or electrostatic interaction. NTFs have been immobilized in hydrogels, microspheres, electrospun nanofibers and combined systems, which serve as depots for sustained local release of protein. These systems generally control drug release by the mechanisms of slow degradation of the materials, slow drug diffusion or a combination of both.
3.1.1.
Hydrogels
Hydrogels have been used in both clinical and basic research to aid in tissue and organ regeneration and engineering. They are water-swollen networks of lightly cross-linked polymer chains, which allow hydrogels to reversibly dehydrate and reswell depending on the environment, thus enabling drug uptake and release [13]. Hydrogels most often used in
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neuroscience application not only have high water content but also fall under the definition of chemically across-linked synthetic hydrogels with viscoelastic properties. A wide range of natural and synthetic hydrogels have been explored for the fabrication of artificial nerve conduits (NC), which is the instrument for repairing damaged peripheral nerves, and some of them have been approved by regulatory authorities for use in human. However, presently marked artificial NC has limited functional capacity for repairing even small-sized nerve gaps. Many researches demonstrated that NTFs play an indispensable role for neuronal survival and axonal regeneration, which is a prerequisite for effective functional re-innervation of severed peripheral nerve. Prolonged delivery of NTFs was also achieved by embedding the drug substances into NC wall, polymeric coating or filled into the lumen of artificial NC. These technologies have been summarized in a recent review paper and will not be discussed here in detail [2]. One limitation of these technologies is the initial burst of NTFs release, as excessive initial NTFs doses can hamper the early axonal regeneration. To minimize the burst release of the NTFs, other sustained strategies including microspheres and electrospun nanofibers have been combined with NC and further developed for localized release of NTFs. This topic will be discussed in later sections. In addition, injectable hydrogel offer considerable promise for controlled and sustained release over time via minimally invasive administration. A variety of materials have been tested as potential vehicles for delivery of NTFs in the treatments of neurodegenerative diseases. For example, injections of an acrylated poly(ethylene glycol) (PEG) plus poly lactic acid (PLA) hydrogel that released NT-3 over a several week period were reported to improve behavior and axon growth after spinal cord injury [14]. In another study, Sofroniew et al. employed an amphiphilic diblock copolypeptide hydrogels (DCH), a highly versatile and finely tunable injectable hydrogels, to form depots that provide sustained delivery of NGF. This proved that DCH can provide a temporary gradient of bioactive protein that is active over a distance of at least several mm in forebrain that is sustained over a subacute time course of at least 4 weeks [15]. Mechanical and physical features which are helpful to consider when constructing a hydrogel include the strength and stiffness, the mesh size and porosity as well as the overall architecture and physical dimensions of the hydrogel. Many of these characteristics can significantly contribute to the effectiveness the hydrogel, from biocompatibility with tissues to how therapeutic agents are released from the hydrogel. In general, fewer cross-links make for softer hydrogels that are ideal for brain and other soft tissues. More specially, the injectable hydrogels with storage modulus (G’) values somewhat lower than that of brain tissue are easily injected, and self-assemble into well-formed deposits of gel networks after injection, with a specifically desired range of brain at just below that of CNS tissues are preferred. Meanwhile, hydrogels has also been investigated for the delivery of NTFs as the treatment of sensorineural hearing loss (SNHL). Havenith et al. applied Gelfoam (a gelatin sponge) infiltrated with BDNF onto the round window membrane (the key barrier separated the middle ear and inner ear) of deafened guinea pigs and evaluated the effect of this
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treatment by structural and functional measures. In the two weeks of Gelfoam BDNF treatment, the survival of spiral ganglion neurons in the inner ear was observed in the low turn of the cochlea, but no significant improvement was observed in the apical turn of the cochlea. This was likely due to such a small amount of drug reaching the apical turn [5]. This issue could be resolved by increasing the residence time of hydrogels on the round window membrane in order to achieve more uniform drug distribution in the inner ear. On the other hand, the cochlear implant (CI) provides electric hearing to patients with certain types of severe hearing loss induced by hair cell loss. Until recently, it was recognized that the process of inserting an electrode deeply into cochlea would destroy all remaining acoustic hearing due to various complications associated with implantation. Hence many researchers tried to find an opportunity to combine the use of drugs with CI to reduce trauma to the inner ear to prevent further degeneration of hearing after implantation [16]. NTFs were also delivered from modified CI by either physically absorbing or entrapment in the coating of CI. Directly absorption of protein on the CI frequently led to the loss of bioactivity and a high initial burst release. Instead, the hydrogel coating of CI provided an alternative to solve this problem. For example, the combination of the conducting polymer poly(3.4-ethylenedioxythiophene) (PEDOT) with an arginineeglycineeaspartic acid (RGD)-modified alginate hydrogel loaded with BDNF created an effective, noncytotoxic, and clinically relevant CI coating. A substantial release of bioactive BDNF into the cochlear fluids within 1 week of implantation with a BDNF-soaked hydrogel-coated implant and this bioactive effect was sustained 2 weeks after implantation. The hydrogel coating of CI was dual-functional: the coating improved the performance of CI, including reduced electrode impedance and improved charge delivery; in addition, the hydrogel also provided the reservoir for bioactive molecules [17].
3.1.2.
Microspheres
An alternative to non-covalent binding is the encapsulation of protein into microspheres, which are often employed as controlled release systems delivered by stereotactic injections to localized disease or injury sites. Many biodegradable synthetic polymers such as poly (lactic-co-glycolic acid) (PLGA) and natural biomaterials have been used to fabricate this type of controlled drug delivery systems. Specific properties of these materials such as the degradation rate can be modified to tailor for particular applications. It has been assumed that a loaded protein drug is released gradually following the polymer degradation kinetics [18]. Natural biomaterials such as collagen, gelatin, chitosan and alginate possess the advantages of excellent protein compatibility and biocompatibility over the synthetic polymers. Moreover, many of them have proved to enhance neural cell adhesion, survival and neurite outgrowth of neurons. While synthetic polymers offer a great choice and flexibility for developing microspheres with customized degradation properties to meet with nerve regeneration rate [2]. An ideal microspheres formulation should have a reasonably high protein encapsulation efficiency, loading capacity, and sustained release of the loaded protein with retained
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bioactivity. The high protein loading and high encapsulation efficiencies are most critical simply due to the extremely high price of therapeutic proteins. Different techniques have been developed to avoid the protein denaturation during the microencapsulation process. Since proteins in the solid state are believed to maintain their bioactivity by drastically reducing conformational mobility in comparison to the large structural change found in the dissolved state, several laboratories are working on protein microencapsulation following S/O/O (solid-in-oil-in-oil) or S/O/W (solid-in-oil-in-water) method. It is, however, noted that dispersing protein particles in organic solvent is not easy. Whichever S/O/O or S/O/W techniques are employed, it is essential to prepare a protein powder with small particle size in the first S/O suspension step. Micronization methods include lyophilization, spray drying, and spray freeze-drying. The complicated micronization step compromises the power of protein protective ability via S/O/O or S/O/W method. Later, the techniques are further modified so that the protein can be firstly dissolved in dimethyl sulfoxide (DMSO) or methanol without inducing the loss of activity. The S/O emulsion is then dispersed into the polymer solution to generate microspheres [18]. Similarly, GDNF was directly suspended in vitamin E (Vit E) and dispersed in PLGA solution to obtain microspheres under very mild conditions (microspheres elaboration method described in Fig. 1). This modified method led to a high microspheres production yield of 76.3 8.4% but a low protein encapsulation efficiency of 27.8 3.1%. The authors observed a burst release of 60% of the loaded GDNF within the first day and then continuous release was reported with almost 100% of the total GDNF recovered at the end of assay (day 133). They also suggested that the protein was released in its bioactive form for more than three months as the evidence of the survival of photoreceptor and retinal ganglion cells in vitro. Furthermore, the intravitreal injection of GDNF/Vit E PLGA microspheres in
the experimental animal model of glaucoma significantly increased the survival of retinal ganglion cells compared to GDNF, Vit E or blank microspheres. The effect was lasted for at least eleven weeks. It was suggested that the formulations prepared might be clinically useful as a neuroprotective tool in the treatment of glaucomatous optic neuropathy [19]. Reducing the initial burst release still remains a challenge when using microspheres. To avoid this issue, some groups have investigated the novel immobilized strategies of proteins in microspheres rather than the absorption of protein on polymer (Fig. 2A). For instance, Yuan and Luo et al. used ionically cross-linked chitosan microspheres fabricated by the emulsion-ionic cross-linking method with sodium tripolyphosphate (STPP) as an ionic cross-linking agent. The NGF encapsulation efficacy ranged from 63% to 88% depending on the concentration of STPP. They reported an initial burst release of 18 to 45% in the first 12 hand the release profiles were also influenced by the concentration of STPP. The higher level of STPP, the slower release of NGF as observed. After the burst stage, the slow release manner was observed over 7 days. However, the total release of encapsulated NGF from the microspheres did not take place at the end of 7 days, indicating NGF retained in the formulations. The result was attributed to the electrostatic interactions between positively NGF (isoelectric points of 9.0e9.35) and negatively charged STPP. In addition, the released NGF was capable of maintaining the viability of PC12 cells, as well as promoting their differentiation [20]. Taken together, electrostatic interaction methods provided potential strategies to immobilize the proteins in the polymers if appropriately designed (Fig. 2B). Another interesting approach to improve the immobilization of NTFs in the microspheres is to apply the natural binding properties of proteins to the component of the extracellular space (ECS) based on their affinity for proteoglycans, such as collagen, or glycosaminoglycans, such as
Fig. 1 e Preparation of protein loaded microspheres by S/O/W emulsion.
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heparin-based delivery system, although these factors do not show a high affinity. As a result of it, a molar excess of immobilized heparin binding was required in the systems to overcome the transient nature of binding interactions and to facilitate effectively sustained release. Mohtaram et al. described the affinity-based delivery systems for NTFs recently [21]. Kraskiewicz et al. created biomimetic hollow microspheres based on collagen to immobilize NGF by using a template method. The extremely high loading efficiency of the reservoir system was proven (90e99%), but the burst release of NGF was observed, as the evidence of about 70 % of total protein released in the first 2 days [22].
3.1.3.
Fig. 2 e Illustration of non-covalent neurotrophic factors binding strategies in microspheres. (A) Adsorption of neurotrophic factors through physicochemical interaction with polymer; (B) Ionic complexation of neurotrophic factors with oppositely charged polymer and the protein release occurs by ion exchange mechanism, which is highly sensitive to the environmental conditions; (C) Affinity-based release of neurotrophic factors from the extracellular matrix which are favorably presented to their receptors.
heparin sulfate (Fig. 2C). The release characteristics depend on the interactions between the drug and matrix in a manner independent of the properties of the matrix itself. The frequently employed affinity-based delivery system is
Electrospun fibers
Electrospinning is a popular technique for preparing tissue engineering scaffolds due to its relative simplicity regarding the generation of fibrous scaffolds with nano- or submicronscale dimensions, which morphologically resembles the natural ECM. Due to the possibility of ultrathin fiber diameters, electrospun fibrous matrices have a large specific surface area which enables effective delivery of biomolecules. Furthermore, the loose bonding between fibers is beneficial for tissue growth and cell migration. These characteristics endue electrospinning with superiority in preparation of bioactive scaffolds. Currently electrospun fibers could be made from a wide range of starting materials, including PLGA, poly(vinyl alcohol)(PVA) and poly (b-caprolactone) (PCL) for encapsulation of growth factors [23]. The easiest way to load biomolecules into electrospun fibers is to dip the scaffolds into an aqueous phase of proteins. With this approach proteins can attach to the scaffolds via physical absorption. Although this approach gives little interference with the activity of loaded biomolecules, it is seldom employed due to the uncontrolled release profiles (Fig. 3A). Another approach to loading proteins approach is the using blend electrospinning, where proteins
Fig. 3 e Fabrication technique of neurotrophic factors loaded electrospun scaffolds. (A) Physical absorption led to fast protein release; (B) Blend electrospinning led to moderate sustained protein release; (C) Instead, coaxial electrospinning might provide proper sustained protein release due to their long diffusion path.
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are mixed within the polymer solution and then used in the electrospinning process to create a hybrid scaffold (Fig. 3B). Valmikinathan et al. employed the blend electrospinning technology to incorporate the bovine serum albumin (BSA)NGF complex into electrospun fibers and their results showed that a sustained release of NGF was obtained for at least 28 days with the relatively lower burst initial release (below 10% of total protein release at the first 1 day) [24]. Coaxial electrospinning, also known as co-electrospinning, is yet another approach to loading biomolecules within fibers. In this approach, two solution (typically polymer solution and biological solution) are coaxially and simultaneously electrospun through different feeding capillary channels in one needle to generate composite fibers with core-shell structures (Fig. 3C). Liu et al. developed a novel composite NC comprised of poly(lactic acid-caprolactone) and NGF by coaxial electrospinning of polymer for the shell and BSA/NGF for the core. Although no in vitro release data of NGF showed, it was concluded that the performance of composited NC was not statistically different from the positive autograft control after sciatic nerve injury, which has provided the clinical “gold standard” for a long period of time in peripheral nerve repair research [25].
3.1.4.
Combined systems
Although hydrogels, microspheres and electrospun fibers are widely used in controlled release systems for the treatment of neurological disorders, obtaining extended, uniform drug release with little initial burst has been challenging [21]. On the other hand, functional recovery from neurological diseases and disorders can only be achieved through the delivery of multiple therapeutic agents, each which requires different time courses of release. For example, GDNF should be released quickly to improve survival during the acute inflammatory response which typically kills the graft within weeks of implantation. Alternatively, BDNF could be released over a period of months to elicit axon extension and tissue development [26]. The different approaches described in this section (hydrogels, microspheres or electrospun fibers) can be combined to obtain above effects. In one example, Han et al. proposed a combined delivery system strategy for sustained release of NGF by the composite comprised of PCL electrospun fiber coupled with poly(ethylene glycol)-poly(b-caprolactone) diacrylate (PEGPCL) hydrogel. The combined delivery system exhibited stable, near-linear, sustained release of the model protein BSA for over two months with a significant reduction in initial burst release, as well as NGF over 28 days in vitro. Bioactivity of NGF was also assessed by examining PC12 cell neurite extension and the results showed the released NGF remained bioactive over 14 days [27]. In another example, PLGA microspheres were encapsulated within degradable poly (ethylene glycol) (PEG)-based hydrogels in a spatial orientation to deliver BDNF or GDNF to brain areas associated with Parkinson’s disease (striatum and substantia nigra, respectively). The ability to control the spatial and temporal release of a therapeutic agent was studied using two preparations of neurotrophic-loaded microspheres incorporated into hydrogel strands designed to release GDNF more quickly and BDNF more slowly (Fig. 4). This would allow GDNF to have a positive impact on transplant survival and BDNF to elicit axon extension over a period of months [26].
Fig. 4 e Schematic showing the hydrogel/microspheres constructs as formed for implantation. Hydrogels were made that encapsulated standard PLGA (rostral, left) and carboxylated PLGA (caudal, right) microspheres into each end. BDNF microspheres were encapsulated and implanted into rostral tissues (targeting striatum) while the GDNF microspheres were oriented in caudal tissues (targeting substantia nigra). The GDNF group had hydrogels containing slow releasing BSA and fast-releasing GDNF. The BDNF group was implanted with hydrogels carrying slow-releasing BDNF and fast-releasing BSA.
3.2.
Covalent neurotrophic factors binding
Covalent immobilization is another strategy for retaining growth factors for longer periods of time at the target site. Covalent immobilization of growth factors was not a previously expected to maintain biological activity because it may negatively affect their binding to the receptors and the subsequent dimerization of the receptors in the plane of the membrane. Nevertheless, if appropriately designed, conjugated growth factors, also called tether growth factors, offer important control over the amount and distribution of these component in solid matrices and facilitate the establishment of growth factor gradients. Chen et al. covalently cross-linked NGF with the glutaraldehyde composite of gelatin-tricalcium phosphate membranes via carbodiimide. After immobilized, NGF can be released from the membrane at least for 60 days and the molecules released from the membrane still remains the bioactivity which promotes the neurite outgrowth of PC12 cell [28].
4.
Challenges and outlook
Over the past two decades, numerous clinical trials involving several different NTFs and neurodegenerative diseases have
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been conducted, and yet the quality and magnitude of any claimed successes do not yet allow one to declare victory [11]. It is because NTFs pose a number of unique and difficult delivery issues. The challenges still exist for further development of NTFs delivery systems includes concerns about (1) protein instability, (2) difficulties in release kinetics control, and (3) unclear therapeutic window.
4.1.
Protein instability
Maintaining protein bioactivity within delivery systems will be essential for further clinical application of NTFs, because the loss of bioactivity of a protein might not only be detrimental to the therapeutic potential, but could also cause immunogenic effects related to exposure of non-native peptide epitopes [29]. Despite the few success stories using various polymers in sustained delivery of NTFs in preclinical research, it needs to be mentioned that the instability of protein during preparation, storage and degradation period is a general problem for polymeric protein delivery system. During the complex preparation process, the growth factor activity might reduce; even through hydrogels delivery systems greatly benefit from their simplicity, conformational changes and denaturation are widespread issues leading to lose of protein activity as well as the irreversible binding of growth factors with polymers, such as PLGA which is attractive for biomolecule delivery because of its tailored degradation rate to achieve controlled release. Although using hydrophilic additives (e.g. PEG composite) was reported to minimize the interaction between protein and polymers, the harmful effect still exist [30]; Protein adsorption and denaturation at the water/solvent interface is one of the major factors for decreased protein bioactivity occurring during the microencapsulation process [18]; In fact, it is well recognized that high voltage and contact with organic solvents during the electrospinning process may be harmful to the growth factor activity [23]. Therefore, optimizing protein stability during the preparation process is a major challenge for effective protein delivery from sustained systems. Currently, there are a few of effective approached to protect growth factors within their delivery systems. One is using the human serum albumin (HSA) or BSA, which occupies the interfaces and shields the therapeutic proteins from contact with hydrophobic surfaces. In addition, they act as proton scavengers during the synthetic polymer degradation process where the acidic microenvironment induced by hydrolysis products of polyesters is also likely to be destructive to growth factor integrity. Some authors used HSA or BSA as protein stabilizer, but they rarely mentioned the effect of applied ratio of the additive to protein (10e50 folds in mass ratio was frequently employed in their work) on growth factor stability [20,27,31]. Meanwhile, a BSA concentration of 0.1% is generally incorporated in most protein release studies in order to keep proteins from aggregation, misfolding and absorption [32,33]. However, 1% BSA in release medium was reported showing a benefit in the recovery of GDNF as compared with 0.1% BSA [19]. Besides, 0.02e0.05% Tween 20 or Tween 40 was frequently used as an additive in the release medium to offer protein protection [22,34,35]. Another approach is adding pH regulators within the delivery systems to maintain pH during
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polymers degradation. The hydrophilic polymer PEG was employed to maintain pH within the delivery systems which functioned as porogen to allow acidic degraded products to rapidly release to outer environment [36], while poorly watersoluble basic salts such as Mg (OH)2 was used to neutralize acidic microenvironment [37]. Moreover, if lyophilization is subjected to be used during the formulation preparation process, lyoprotectants like sucrose and dextran within formulations might avoid the protein from degradation [23].
4.2.
Difficulties in release kinetics control
The biological effects of NTFs depend on the precise delivery paradigm. Inadequate NTFs dose and release kinetics often led to aberrant axonal growth, which cannot be improved by further NTFs delivery [2]. Instead, the initial burst release of NTFs results in inhabitation of the early axonal regeneration. Optimal NTFs release kinetics must be compatible with axonal growth and regeneration, which means that NTFs released must be in keeping with physiological nerve and reinnervated organ regeneration [2]. However, the exact kinetics required for therapeutic benefit is currently not clear and likely varies between situations [11]. A further complication for treating neurodegenerative diseases is that adequate levels of NTFs must be maintained for very long period of time (i.e., for years). Unfortunately, traditional pharmaceutical formulations and delivery approaches are unable to solve this problem. Nevertheless, the sustained delivery of NTFs also provides a potential for subsidiary nerve protection over short period (i.e., for months), which is regarded as key step before further medical treatments. A very effective means to improve functional axonal regeneration is by controlled delivery of multiple NTFs with their individually optimal kinetics [2,26]. To couple with the optimal kinetics for two or more proteins, suitable delivery systems must be developed to enable more efficient release kinetics. To overcome the long-standing delivery issues, gene transfer has emerged as a practical means of potentially providing the ‘enabling’ technology required for translating the use of these proteins into viable biotherapeutics for neurodegenerative diseases [11]. In addition, it is widely accepted that Schwann cells play an indispensable role in axonal regeneration. Therefore, efficient use of such glial cells at appropriate sites may improve regenerative efficacy, despite the fact that the isolation and culturing of persynaptic Schwann cells on large scale may present new challenges [38]. As a result of this, stem cells, like adipose-derived stem cells, have been proposed as alternative to obtain Schwann cells [39,40]. Although gene transfer and cells transplantation have shown mediate beneficial effects on nerve regeneration, clinically relevant protocols on safe and efficient use of them are yet to be developed.
4.3.
Unclear therapeutic window
The effect of NTFs are dose-dependent [11]. Currently, there is no available data concerning the therapeutic concentration of NTFs in targeted tissues or organs that can be used to evaluate the efficient therapeutic effect. To define the therapeutic window of NTFs, pertinent evaluation approaches should be
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developed. Ideally, the evaluation approaches should probably be practiced in scale and then the complicated biological effect would be assayed widely. Because of this, in vitro bioassay of NTFs is preferred, including the employment of PC12 cells and chicken embryonic dorsal root ganglia (DRG) methods. PC12 cells have been widely used as cell models for neuronal differentiation in response to exogenous signals, such as NTFs and neurotransmitters [41]. Previous reports demonstrated that effective NGF to stimulate PC12 cell neurite outgrowth is in the range of 0.1e50 ng/ml [42]. Another investigators applied DRG bioassay to optimize axonal growth response of NTFs and the results showed that the ideally dose was observed at 1e10 ng/ml of GDNF or NGF when used as single factors, whereas the optimal dose range was 0.1e1 ng/ ml for combined GDNF and NGF [9]. However, in vitro data should be ‘translated’ to in vivo information before clinical application, which met with enormous challenge. Some complications in a human clinical setting include genetic background, lifestyle, physical activity and age of the patients as well as variable pathology and additional medications, which all may affect the required dose. In conclusion, successful NTFs delivery requires dosage customization for each factor and delivery system, in both preclinical model and clinical case.
5.
Conclusion and summary
The reliable and meaningful clinical benefits of NTFs will intimately depend on their optimal localized delivery in a given context. A delivery system designed for nerve regeneration should (ⅰ) provide a time and dose-controlled release of the bioactive NTFs and (ⅱ) offer multiple NTFs delivery with individually optimal kinetics to achieve multi-functions (e.g. prevention of neuronal death and promotion of axonal growth). Additional requirements for a carrier include high biocompatibility, adequate biodegradability, ease of manufacturing (reproducibility and scale up), and cost effectiveness. Particular challenge for developing an optimal delivery systems for NTFs encompass the achievement of maintain optimal protein retention over a long period of time in target tissues. Optimizing and customizing sustained delivery systems of NTFs require continued attention but will hopefully lead to more efficient therapies for neurological disorders.
Acknowledgment This study was financially supported by the National Natural Science Foundation of China (Grant No. 81102401). We thank Xian Xu for editing the manuscript.
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[2] Madduri S, Gander B. Growth factor delivery systems and repair strategies for damaged peripheral nerves. J Control Release 2012;161:274e282. [3] Johnson TV, Bull ND, Martin KR. Neurotrophic factor delivery as a protective treatment for glaucoma. Exp Eye Res 2011;93:196e203. [4] Budenz CL, Pfingst BE, Raphael Y. The use of neurotrophin therapy in the inner ear to augment cochlear implantation outcomes. Anat Rec (Hoboken) 2012;295:1896e1908. [5] Havenith S, Versnel H, Agterberg MJ, et al. Spiral ganglion cell survival after round window membrane application of brainderived neurotrophic factor using gelfoam as carrier. Hear Res 2011;272:168e177. [6] Aloe L, Rocco ML, Bianchi P, et al. Nerve growth factor: from the early discoveries to the potential clinical use. J Transl Med 2012;10:239. [7] Manni L, Rocco ML, Bianchi P, et al. Nerve growth factor: basic studies and possible therapeutic applications. Growth Factors 2013;31:115e122. [8] Rich KM, Luszczynski JR, Osborne PA, et al. Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury. J Neurocytol 1987;16:261e268. [9] Madduri S, Papaloizos M, Gander B. Synergistic effect of GDNF and NGF on axonal branching and elongation in vitro. Neurosci Res 2009;65:88e97. [10] Madduri S, di Summa P, Papaloizos M, et al. Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats. Biomaterials 2010;31:8402e8409. [11] Bartus RT. Translating the therapeutic potential of neurotrophic factors to clinical ’proof of concept’: a personal saga achieving a career-long quest. Neurobiol Dis 2012;48:153e178. [12] Capsoni S, Marinelli S, Ceci M, et al. Intranasal “painless” human nerve growth factors slows amyloid neurodegeneration and prevents memory deficits in App X PS1 mice. PLoS One 2012;7:e37555. [13] Aurand ER, Lampe KJ, Bjugstad KB. Defining and designing polymers and hydrogels for neural tissue engineering. Neurosci Res 2012;72:199e213. [14] Piantino J, Burdick JA, Goldberg D, et al. An injectable, biodegradable hydrogel for trophic factor delivery enhances axonal rewiring and improves performance after spinal cord injury. Exp Neurol 2006;201:359e367. [15] Song B, Song J, Zhang S, et al. Sustained local delivery of bioactive nerve growth factor in the central nervous system via tunable diblock copolypeptide hydrogel depots. Biomaterials 2012;33:9105e9116. [16] Staecker H, Jolly C, Garnham C. Cochlear implantation: an opportunity for drug development. Drug Discov Today 2010;15:314e321. [17] Chikar JA, Hendricks JL, Richardson-Burns SM, et al. The use of a dual PEDOT and RGD-functionalized alginate hydrogel coating to provide sustained drug delivery and improved cochlear implant function. Biomaterials 2012;33:1982e1990. [18] Ye M, Kim S, Park K. Issues in long-term protein delivery using biodegradable microparticles. J Control Release 2010;146:241e260. [19] Checa-Casalengua P, Jiang C, Bravo-Osuna I, et al. Retinal ganglion cells survival in a glaucoma model by GDNF/Vit E PLGA microspheres prepared according to a novel microencapsulation procedure. J Control Release 2011;156:92e100. [20] Zeng W, Huang J, Hu X, et al. Ionically cross-linked chitosan microspheres for controlled release of bioactive nerve growth factor. Int J Pharm 2011;421:283e290.
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[21] Mohtaram NK, Montgomery A, Willerth SM. Biomaterialbased drug delivery systems for the controlled release of neurotrophic factors. Biomed Mater 2013;8:022001. [22] Kraskiewicz H, Breen B, Sargeant T, et al. Assembly of protein-based hollow spheres encapsulating a therapeutic factor. ACS Chem Neurosci 2013;4:1297e1304. [23] Ji W, Sun Y, Yang F, et al. Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications. Pharm Res 2011;28:1259e1272. [24] Valmikinathan CM, Defroda S, Yu X. Polycaprolactone and bovine serum albumin based nanofibers for controlled release of nerve growth factor. Biomacromolecules 2009;10:1084e1089. [25] Liu JJ, Wang CY, Wang JG, et al. Peripheral nerve regeneration using composite poly(lactic acid-caprolactone)/nerve growth factor conduits prepared by coaxial electrospinning. J Biomed Mater Res A 2011;96:13e20. [26] Lampe KJ, Kern DS, Mahoney MJ, et al. The administration of BDNF and GDNF to the brain via PLGA microparticles patterned within a degradable PEG-based hydrogel: protein distribution and the glial response. J Biomed Mater Res A 2011;96:595e607. [27] Han N, Johnson J, Lannutti JJ, et al. Hydrogel-electrospun fiber composite materials for hydrophilic protein release. J Control Release 2012;158:165e170. [28] Chen PR, Chen MH, Lin FH, et al. Release characteristics and bioactivity of gelatin-tricalcium phosphate membranes covalently immobilized with nerve growth factors. Biomaterials 2005;26:6579e6587. [29] van der Walle CF, Sharma G, Ravi Kumar M. Current approaches to stabilising and analysing proteins during microencapsulation in PLGA. Expert Opin Drug Deliv 2009;6:177e186. [30] Johnson PJ, Skornia SL, Stabenfeldt SE, et al. Maintaining bioactivity of NGF for controlled release from PLGA using PEG. J Biomed Mater Res A 2008;86:420e427. [31] Xu X, Yu H, Gao S, et al. Polyphosphoester microspheres for sustained release of biologically active nerve growth factor. Biomaterials 2002;23:3765e3772.
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Review
Current sustained delivery strategies for the design of local neurotrophic factors in treatment of neurological disorders Hongzhuo Liu a, Yanyan Zhou a, Shichao Chen b, Meng Bu c, Jiayu Xin c, Sanming Li a,* a
School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, China School of Life Science & Biopharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China c Harbin Medical University, Harbin 150081, China b
article info
abstract
Article history:
Although therapeutic potential of neurotrophic factors (NTFs) has been well recognized for
Received 19 August 2013
over two decades, attempts to translate that potential to the clinic have been disap-
Received in revised form
pointing, largely due to significant obstacles in delivery, including inadequate protein dose/
8 October 2013
kinetics released at target sites. Considerable efforts have been made to improve the
Accepted 9 October 2013
therapeutic performance of NTFs. This articles reviews recent developments in localized delivery systems of NTFs for the neurological disorders treatments with a main focus on sustained delivery strategies. Different non-covalent binding approaches have been
Keywords:
employed to immobilize proteins in hydrogels, microspheres, electrospun nanofibers, and
Neurotrophic factors
their combined systems, which serve as depots for sustained local release of NTFs. The
Neurological disorders
challenges associated with current NTFs delivery systems and how these systems can be
Drug delivery
applied to neurological diseases and disorders have been discussed in the review. In
Sustained delivery
conclusion, optimal delivery systems for NTFs will be needed for reliable and meaningful
Release kinetics
clinical benefits; ideally, delivering a time and dose-controlled release of bioactive multiNTFs at different individual optimal kinetics to achieve multi-functions in target tissues is significant preferred. ª 2013 Shenyang Pharmaceutical University. Production and hosting by Elsevier B.V. All rights reserved.
1.
Introduction
Neurotrophic factors are polypeptides primarily known to regulate the survival and differentiation of nerve cells during the development of the peripheral and central nervous
systems. Over the past two decades, scientists have had great expectations for NTFs in the treatment of neurological disorders, including the most common central neurodegenerative diseases, Alzheimer’s (AD) and Parkinson’s (PD) [1]. This approach may also prove effective for the peripheral nerve
* Corresponding author. Tel.: þ86 24 23986258; fax: þ86 24 23986293. E-mail address: [email protected] (S. Li). Peer review under responsibility of Shenyang Pharmaceutical University
Production and hosting by Elsevier 1818-0876/$ e see front matter ª 2013 Shenyang Pharmaceutical University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ajps.2013.10.003
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injury, which is a serious issue affecting 2.8% of trauma patients and many of whom will be subjected to life-long disability [2]. Moreover, NTFs also play an important role in the development of potential neuroprotective glaucoma and hearing disorders treatments [3e5]. Despite their obvious attractiveness as therapeutic agents, they have some serious restrictions in clinical use. The most limiting of these is their short biological half-life and vulnerability to structural disruption or modification, leading to loss of bioactivity. Therefore, protein drug delivery systems are needed not only to improve the biological utilization by sustained release of bioactive NTFs to target site at sufficient concentration, but also to protect their bioactivity from degradation by direct exposure to harsh environments. There has been a growing interest to develop new strategies for effective delivery of NTFs. This article reviews some widely investigated NTFs and their delivery systems, summarizes the difficulties in their neurodegenerative diseases therapy and the most pertinent evaluation system for testing delivery systems, and finally provides perspectives for advanced nerve repair strategies that may hold promise for enhancing therapeutic efficacy.
2.
Neurotrophic factor
Nerve growth factor (NGF), discovered by R. Levi Montalcini almost 60 years ago, is regarded as the first discovered and as an important member of neurotrophin family [6]. Later, three NGF homologous growth factors were identified: the brainderived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4/5. Each has its distinct and/or overlapping activities within the developing peripheral and central nervous systems and are collectively indicated as neurotrophins [7]. Widely studied NTFs also include the glial cell line-derived neurotrophic factor (GDNF, belonging to the transforming growth factor-b superfamily), the glial growth factor (GGF) (a member of the neuregulin family), neuropoietic cytokines [such as ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF)] and other cytokines [2]. Since important NTFs in adulthood as well as development, reducing the level of one or more of these proteins may be responsible for the neurological disorders. Each of the factors has been employed in the neurorestorative therapy in some form or another in clinical trials. To brief illustrate, neurotrophin family and GDNF are addressed when selecting or designing a delivery system in this review. Although GDNF shows only limited amino-acid sequence homology with the member of neurotrophins family, it has somewhat conformational similarity as they all have the characteristic cystine knot structure motif with the formation of three disulphide bonds. The above proteins differ in their spectrum of action when applied as a single factor. NGF is essential for the development and phenotypic maintenance of neurons in the peripheral nervous system (PNS) and for the functional integrity of cholinergic neurons in the central nervous system (CNS) [1]. It has been considered as a very powerful and selective growth factor for sympathetic and sensory neurons, especially primarily survival and axonal outgrowth of sensory neurons [8]. Meanwhile BDNF supports the survival and maintenance of sensory neurons, retinal
ganglia, certain cholinergic neurons, spinal motor neurons and some dopaminergic neurons [1]. NT-3 helps to support the survival and differentiation of existing neurons, and also encourages the growth and differentiation of new neurons and new neurons and synapses [1]. GDNF shows pronounced effects on the survival of midbrain dopaminergic neurons. Even more interesting, it is identified to be key factor for motor axonal regeneration for its ability to promoting axonal elongation [9,10]. Neurotrophin family binds to two different receptors: high affinity binding via tropomyosin receptor kinase (Trk)-A, B or C and low affinity nerve growth factor receptor (p75 neurotrophin receptor, P75NTR). NGF binds TrkA; BDNF, NT-4/5 binds TrkB; NT-3 binds TrkC. Finally, the binding of GDNF by its homodimeric receptor tyrosine kinase complex leads to activation of the TrkA pathway, which in the case of GDNF is highly complex and interdependent [1].
3. Current delivery strategies for neurotrophic factors Though applied to a wide spectrum of neurological diseases, the clinical utilization of NTFs, , remains limitations by important adverse events induced by exposing non-targeted tissues [11] coupled with the mild side effects like those derived from the effects of NGF on pain system [12]. These effects are especially when systemically administration. Moreover, NTFs undergo very short biological half-life in circulation and rapid degradation in vivo, and also present poor permeability across the biological barriers [11]. Therefore, to an efficacious clinical outcome, localized delivery for NTFs is preferred. Nevertheless, the clinical outcome remained, however generally unsatisfactory, and was probably due to inadequate NTFs doses and/or release kinetics at the optimal location, undesired initial burst release, and the use of single NTFs rather than multiple factors as occurs naturally [2]. There have been many attempts to deliver various NTFs to target sites by using diverse types of synthetic and natural materials in order to modulate the release kinetics. Generally, the proteins were designed to be immobilized in the systems through non-covalent or covalent means.
3.1.
Non-covalent neurotrophic factors binding
Non-covalent binding includes physical entrapment, adsorption or electrostatic interaction. NTFs have been immobilized in hydrogels, microspheres, electrospun nanofibers and combined systems, which serve as depots for sustained local release of protein. These systems generally control drug release by the mechanisms of slow degradation of the materials, slow drug diffusion or a combination of both.
3.1.1.
Hydrogels
Hydrogels have been used in both clinical and basic research to aid in tissue and organ regeneration and engineering. They are water-swollen networks of lightly cross-linked polymer chains, which allow hydrogels to reversibly dehydrate and reswell depending on the environment, thus enabling drug uptake and release [13]. Hydrogels most often used in
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neuroscience application not only have high water content but also fall under the definition of chemically across-linked synthetic hydrogels with viscoelastic properties. A wide range of natural and synthetic hydrogels have been explored for the fabrication of artificial nerve conduits (NC), which is the instrument for repairing damaged peripheral nerves, and some of them have been approved by regulatory authorities for use in human. However, presently marked artificial NC has limited functional capacity for repairing even small-sized nerve gaps. Many researches demonstrated that NTFs play an indispensable role for neuronal survival and axonal regeneration, which is a prerequisite for effective functional re-innervation of severed peripheral nerve. Prolonged delivery of NTFs was also achieved by embedding the drug substances into NC wall, polymeric coating or filled into the lumen of artificial NC. These technologies have been summarized in a recent review paper and will not be discussed here in detail [2]. One limitation of these technologies is the initial burst of NTFs release, as excessive initial NTFs doses can hamper the early axonal regeneration. To minimize the burst release of the NTFs, other sustained strategies including microspheres and electrospun nanofibers have been combined with NC and further developed for localized release of NTFs. This topic will be discussed in later sections. In addition, injectable hydrogel offer considerable promise for controlled and sustained release over time via minimally invasive administration. A variety of materials have been tested as potential vehicles for delivery of NTFs in the treatments of neurodegenerative diseases. For example, injections of an acrylated poly(ethylene glycol) (PEG) plus poly lactic acid (PLA) hydrogel that released NT-3 over a several week period were reported to improve behavior and axon growth after spinal cord injury [14]. In another study, Sofroniew et al. employed an amphiphilic diblock copolypeptide hydrogels (DCH), a highly versatile and finely tunable injectable hydrogels, to form depots that provide sustained delivery of NGF. This proved that DCH can provide a temporary gradient of bioactive protein that is active over a distance of at least several mm in forebrain that is sustained over a subacute time course of at least 4 weeks [15]. Mechanical and physical features which are helpful to consider when constructing a hydrogel include the strength and stiffness, the mesh size and porosity as well as the overall architecture and physical dimensions of the hydrogel. Many of these characteristics can significantly contribute to the effectiveness the hydrogel, from biocompatibility with tissues to how therapeutic agents are released from the hydrogel. In general, fewer cross-links make for softer hydrogels that are ideal for brain and other soft tissues. More specially, the injectable hydrogels with storage modulus (G’) values somewhat lower than that of brain tissue are easily injected, and self-assemble into well-formed deposits of gel networks after injection, with a specifically desired range of brain at just below that of CNS tissues are preferred. Meanwhile, hydrogels has also been investigated for the delivery of NTFs as the treatment of sensorineural hearing loss (SNHL). Havenith et al. applied Gelfoam (a gelatin sponge) infiltrated with BDNF onto the round window membrane (the key barrier separated the middle ear and inner ear) of deafened guinea pigs and evaluated the effect of this
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treatment by structural and functional measures. In the two weeks of Gelfoam BDNF treatment, the survival of spiral ganglion neurons in the inner ear was observed in the low turn of the cochlea, but no significant improvement was observed in the apical turn of the cochlea. This was likely due to such a small amount of drug reaching the apical turn [5]. This issue could be resolved by increasing the residence time of hydrogels on the round window membrane in order to achieve more uniform drug distribution in the inner ear. On the other hand, the cochlear implant (CI) provides electric hearing to patients with certain types of severe hearing loss induced by hair cell loss. Until recently, it was recognized that the process of inserting an electrode deeply into cochlea would destroy all remaining acoustic hearing due to various complications associated with implantation. Hence many researchers tried to find an opportunity to combine the use of drugs with CI to reduce trauma to the inner ear to prevent further degeneration of hearing after implantation [16]. NTFs were also delivered from modified CI by either physically absorbing or entrapment in the coating of CI. Directly absorption of protein on the CI frequently led to the loss of bioactivity and a high initial burst release. Instead, the hydrogel coating of CI provided an alternative to solve this problem. For example, the combination of the conducting polymer poly(3.4-ethylenedioxythiophene) (PEDOT) with an arginineeglycineeaspartic acid (RGD)-modified alginate hydrogel loaded with BDNF created an effective, noncytotoxic, and clinically relevant CI coating. A substantial release of bioactive BDNF into the cochlear fluids within 1 week of implantation with a BDNF-soaked hydrogel-coated implant and this bioactive effect was sustained 2 weeks after implantation. The hydrogel coating of CI was dual-functional: the coating improved the performance of CI, including reduced electrode impedance and improved charge delivery; in addition, the hydrogel also provided the reservoir for bioactive molecules [17].
3.1.2.
Microspheres
An alternative to non-covalent binding is the encapsulation of protein into microspheres, which are often employed as controlled release systems delivered by stereotactic injections to localized disease or injury sites. Many biodegradable synthetic polymers such as poly (lactic-co-glycolic acid) (PLGA) and natural biomaterials have been used to fabricate this type of controlled drug delivery systems. Specific properties of these materials such as the degradation rate can be modified to tailor for particular applications. It has been assumed that a loaded protein drug is released gradually following the polymer degradation kinetics [18]. Natural biomaterials such as collagen, gelatin, chitosan and alginate possess the advantages of excellent protein compatibility and biocompatibility over the synthetic polymers. Moreover, many of them have proved to enhance neural cell adhesion, survival and neurite outgrowth of neurons. While synthetic polymers offer a great choice and flexibility for developing microspheres with customized degradation properties to meet with nerve regeneration rate [2]. An ideal microspheres formulation should have a reasonably high protein encapsulation efficiency, loading capacity, and sustained release of the loaded protein with retained
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bioactivity. The high protein loading and high encapsulation efficiencies are most critical simply due to the extremely high price of therapeutic proteins. Different techniques have been developed to avoid the protein denaturation during the microencapsulation process. Since proteins in the solid state are believed to maintain their bioactivity by drastically reducing conformational mobility in comparison to the large structural change found in the dissolved state, several laboratories are working on protein microencapsulation following S/O/O (solid-in-oil-in-oil) or S/O/W (solid-in-oil-in-water) method. It is, however, noted that dispersing protein particles in organic solvent is not easy. Whichever S/O/O or S/O/W techniques are employed, it is essential to prepare a protein powder with small particle size in the first S/O suspension step. Micronization methods include lyophilization, spray drying, and spray freeze-drying. The complicated micronization step compromises the power of protein protective ability via S/O/O or S/O/W method. Later, the techniques are further modified so that the protein can be firstly dissolved in dimethyl sulfoxide (DMSO) or methanol without inducing the loss of activity. The S/O emulsion is then dispersed into the polymer solution to generate microspheres [18]. Similarly, GDNF was directly suspended in vitamin E (Vit E) and dispersed in PLGA solution to obtain microspheres under very mild conditions (microspheres elaboration method described in Fig. 1). This modified method led to a high microspheres production yield of 76.3 8.4% but a low protein encapsulation efficiency of 27.8 3.1%. The authors observed a burst release of 60% of the loaded GDNF within the first day and then continuous release was reported with almost 100% of the total GDNF recovered at the end of assay (day 133). They also suggested that the protein was released in its bioactive form for more than three months as the evidence of the survival of photoreceptor and retinal ganglion cells in vitro. Furthermore, the intravitreal injection of GDNF/Vit E PLGA microspheres in
the experimental animal model of glaucoma significantly increased the survival of retinal ganglion cells compared to GDNF, Vit E or blank microspheres. The effect was lasted for at least eleven weeks. It was suggested that the formulations prepared might be clinically useful as a neuroprotective tool in the treatment of glaucomatous optic neuropathy [19]. Reducing the initial burst release still remains a challenge when using microspheres. To avoid this issue, some groups have investigated the novel immobilized strategies of proteins in microspheres rather than the absorption of protein on polymer (Fig. 2A). For instance, Yuan and Luo et al. used ionically cross-linked chitosan microspheres fabricated by the emulsion-ionic cross-linking method with sodium tripolyphosphate (STPP) as an ionic cross-linking agent. The NGF encapsulation efficacy ranged from 63% to 88% depending on the concentration of STPP. They reported an initial burst release of 18 to 45% in the first 12 hand the release profiles were also influenced by the concentration of STPP. The higher level of STPP, the slower release of NGF as observed. After the burst stage, the slow release manner was observed over 7 days. However, the total release of encapsulated NGF from the microspheres did not take place at the end of 7 days, indicating NGF retained in the formulations. The result was attributed to the electrostatic interactions between positively NGF (isoelectric points of 9.0e9.35) and negatively charged STPP. In addition, the released NGF was capable of maintaining the viability of PC12 cells, as well as promoting their differentiation [20]. Taken together, electrostatic interaction methods provided potential strategies to immobilize the proteins in the polymers if appropriately designed (Fig. 2B). Another interesting approach to improve the immobilization of NTFs in the microspheres is to apply the natural binding properties of proteins to the component of the extracellular space (ECS) based on their affinity for proteoglycans, such as collagen, or glycosaminoglycans, such as
Fig. 1 e Preparation of protein loaded microspheres by S/O/W emulsion.
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heparin-based delivery system, although these factors do not show a high affinity. As a result of it, a molar excess of immobilized heparin binding was required in the systems to overcome the transient nature of binding interactions and to facilitate effectively sustained release. Mohtaram et al. described the affinity-based delivery systems for NTFs recently [21]. Kraskiewicz et al. created biomimetic hollow microspheres based on collagen to immobilize NGF by using a template method. The extremely high loading efficiency of the reservoir system was proven (90e99%), but the burst release of NGF was observed, as the evidence of about 70 % of total protein released in the first 2 days [22].
3.1.3.
Fig. 2 e Illustration of non-covalent neurotrophic factors binding strategies in microspheres. (A) Adsorption of neurotrophic factors through physicochemical interaction with polymer; (B) Ionic complexation of neurotrophic factors with oppositely charged polymer and the protein release occurs by ion exchange mechanism, which is highly sensitive to the environmental conditions; (C) Affinity-based release of neurotrophic factors from the extracellular matrix which are favorably presented to their receptors.
heparin sulfate (Fig. 2C). The release characteristics depend on the interactions between the drug and matrix in a manner independent of the properties of the matrix itself. The frequently employed affinity-based delivery system is
Electrospun fibers
Electrospinning is a popular technique for preparing tissue engineering scaffolds due to its relative simplicity regarding the generation of fibrous scaffolds with nano- or submicronscale dimensions, which morphologically resembles the natural ECM. Due to the possibility of ultrathin fiber diameters, electrospun fibrous matrices have a large specific surface area which enables effective delivery of biomolecules. Furthermore, the loose bonding between fibers is beneficial for tissue growth and cell migration. These characteristics endue electrospinning with superiority in preparation of bioactive scaffolds. Currently electrospun fibers could be made from a wide range of starting materials, including PLGA, poly(vinyl alcohol)(PVA) and poly (b-caprolactone) (PCL) for encapsulation of growth factors [23]. The easiest way to load biomolecules into electrospun fibers is to dip the scaffolds into an aqueous phase of proteins. With this approach proteins can attach to the scaffolds via physical absorption. Although this approach gives little interference with the activity of loaded biomolecules, it is seldom employed due to the uncontrolled release profiles (Fig. 3A). Another approach to loading proteins approach is the using blend electrospinning, where proteins
Fig. 3 e Fabrication technique of neurotrophic factors loaded electrospun scaffolds. (A) Physical absorption led to fast protein release; (B) Blend electrospinning led to moderate sustained protein release; (C) Instead, coaxial electrospinning might provide proper sustained protein release due to their long diffusion path.
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are mixed within the polymer solution and then used in the electrospinning process to create a hybrid scaffold (Fig. 3B). Valmikinathan et al. employed the blend electrospinning technology to incorporate the bovine serum albumin (BSA)NGF complex into electrospun fibers and their results showed that a sustained release of NGF was obtained for at least 28 days with the relatively lower burst initial release (below 10% of total protein release at the first 1 day) [24]. Coaxial electrospinning, also known as co-electrospinning, is yet another approach to loading biomolecules within fibers. In this approach, two solution (typically polymer solution and biological solution) are coaxially and simultaneously electrospun through different feeding capillary channels in one needle to generate composite fibers with core-shell structures (Fig. 3C). Liu et al. developed a novel composite NC comprised of poly(lactic acid-caprolactone) and NGF by coaxial electrospinning of polymer for the shell and BSA/NGF for the core. Although no in vitro release data of NGF showed, it was concluded that the performance of composited NC was not statistically different from the positive autograft control after sciatic nerve injury, which has provided the clinical “gold standard” for a long period of time in peripheral nerve repair research [25].
3.1.4.
Combined systems
Although hydrogels, microspheres and electrospun fibers are widely used in controlled release systems for the treatment of neurological disorders, obtaining extended, uniform drug release with little initial burst has been challenging [21]. On the other hand, functional recovery from neurological diseases and disorders can only be achieved through the delivery of multiple therapeutic agents, each which requires different time courses of release. For example, GDNF should be released quickly to improve survival during the acute inflammatory response which typically kills the graft within weeks of implantation. Alternatively, BDNF could be released over a period of months to elicit axon extension and tissue development [26]. The different approaches described in this section (hydrogels, microspheres or electrospun fibers) can be combined to obtain above effects. In one example, Han et al. proposed a combined delivery system strategy for sustained release of NGF by the composite comprised of PCL electrospun fiber coupled with poly(ethylene glycol)-poly(b-caprolactone) diacrylate (PEGPCL) hydrogel. The combined delivery system exhibited stable, near-linear, sustained release of the model protein BSA for over two months with a significant reduction in initial burst release, as well as NGF over 28 days in vitro. Bioactivity of NGF was also assessed by examining PC12 cell neurite extension and the results showed the released NGF remained bioactive over 14 days [27]. In another example, PLGA microspheres were encapsulated within degradable poly (ethylene glycol) (PEG)-based hydrogels in a spatial orientation to deliver BDNF or GDNF to brain areas associated with Parkinson’s disease (striatum and substantia nigra, respectively). The ability to control the spatial and temporal release of a therapeutic agent was studied using two preparations of neurotrophic-loaded microspheres incorporated into hydrogel strands designed to release GDNF more quickly and BDNF more slowly (Fig. 4). This would allow GDNF to have a positive impact on transplant survival and BDNF to elicit axon extension over a period of months [26].
Fig. 4 e Schematic showing the hydrogel/microspheres constructs as formed for implantation. Hydrogels were made that encapsulated standard PLGA (rostral, left) and carboxylated PLGA (caudal, right) microspheres into each end. BDNF microspheres were encapsulated and implanted into rostral tissues (targeting striatum) while the GDNF microspheres were oriented in caudal tissues (targeting substantia nigra). The GDNF group had hydrogels containing slow releasing BSA and fast-releasing GDNF. The BDNF group was implanted with hydrogels carrying slow-releasing BDNF and fast-releasing BSA.
3.2.
Covalent neurotrophic factors binding
Covalent immobilization is another strategy for retaining growth factors for longer periods of time at the target site. Covalent immobilization of growth factors was not a previously expected to maintain biological activity because it may negatively affect their binding to the receptors and the subsequent dimerization of the receptors in the plane of the membrane. Nevertheless, if appropriately designed, conjugated growth factors, also called tether growth factors, offer important control over the amount and distribution of these component in solid matrices and facilitate the establishment of growth factor gradients. Chen et al. covalently cross-linked NGF with the glutaraldehyde composite of gelatin-tricalcium phosphate membranes via carbodiimide. After immobilized, NGF can be released from the membrane at least for 60 days and the molecules released from the membrane still remains the bioactivity which promotes the neurite outgrowth of PC12 cell [28].
4.
Challenges and outlook
Over the past two decades, numerous clinical trials involving several different NTFs and neurodegenerative diseases have
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been conducted, and yet the quality and magnitude of any claimed successes do not yet allow one to declare victory [11]. It is because NTFs pose a number of unique and difficult delivery issues. The challenges still exist for further development of NTFs delivery systems includes concerns about (1) protein instability, (2) difficulties in release kinetics control, and (3) unclear therapeutic window.
4.1.
Protein instability
Maintaining protein bioactivity within delivery systems will be essential for further clinical application of NTFs, because the loss of bioactivity of a protein might not only be detrimental to the therapeutic potential, but could also cause immunogenic effects related to exposure of non-native peptide epitopes [29]. Despite the few success stories using various polymers in sustained delivery of NTFs in preclinical research, it needs to be mentioned that the instability of protein during preparation, storage and degradation period is a general problem for polymeric protein delivery system. During the complex preparation process, the growth factor activity might reduce; even through hydrogels delivery systems greatly benefit from their simplicity, conformational changes and denaturation are widespread issues leading to lose of protein activity as well as the irreversible binding of growth factors with polymers, such as PLGA which is attractive for biomolecule delivery because of its tailored degradation rate to achieve controlled release. Although using hydrophilic additives (e.g. PEG composite) was reported to minimize the interaction between protein and polymers, the harmful effect still exist [30]; Protein adsorption and denaturation at the water/solvent interface is one of the major factors for decreased protein bioactivity occurring during the microencapsulation process [18]; In fact, it is well recognized that high voltage and contact with organic solvents during the electrospinning process may be harmful to the growth factor activity [23]. Therefore, optimizing protein stability during the preparation process is a major challenge for effective protein delivery from sustained systems. Currently, there are a few of effective approached to protect growth factors within their delivery systems. One is using the human serum albumin (HSA) or BSA, which occupies the interfaces and shields the therapeutic proteins from contact with hydrophobic surfaces. In addition, they act as proton scavengers during the synthetic polymer degradation process where the acidic microenvironment induced by hydrolysis products of polyesters is also likely to be destructive to growth factor integrity. Some authors used HSA or BSA as protein stabilizer, but they rarely mentioned the effect of applied ratio of the additive to protein (10e50 folds in mass ratio was frequently employed in their work) on growth factor stability [20,27,31]. Meanwhile, a BSA concentration of 0.1% is generally incorporated in most protein release studies in order to keep proteins from aggregation, misfolding and absorption [32,33]. However, 1% BSA in release medium was reported showing a benefit in the recovery of GDNF as compared with 0.1% BSA [19]. Besides, 0.02e0.05% Tween 20 or Tween 40 was frequently used as an additive in the release medium to offer protein protection [22,34,35]. Another approach is adding pH regulators within the delivery systems to maintain pH during
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polymers degradation. The hydrophilic polymer PEG was employed to maintain pH within the delivery systems which functioned as porogen to allow acidic degraded products to rapidly release to outer environment [36], while poorly watersoluble basic salts such as Mg (OH)2 was used to neutralize acidic microenvironment [37]. Moreover, if lyophilization is subjected to be used during the formulation preparation process, lyoprotectants like sucrose and dextran within formulations might avoid the protein from degradation [23].
4.2.
Difficulties in release kinetics control
The biological effects of NTFs depend on the precise delivery paradigm. Inadequate NTFs dose and release kinetics often led to aberrant axonal growth, which cannot be improved by further NTFs delivery [2]. Instead, the initial burst release of NTFs results in inhabitation of the early axonal regeneration. Optimal NTFs release kinetics must be compatible with axonal growth and regeneration, which means that NTFs released must be in keeping with physiological nerve and reinnervated organ regeneration [2]. However, the exact kinetics required for therapeutic benefit is currently not clear and likely varies between situations [11]. A further complication for treating neurodegenerative diseases is that adequate levels of NTFs must be maintained for very long period of time (i.e., for years). Unfortunately, traditional pharmaceutical formulations and delivery approaches are unable to solve this problem. Nevertheless, the sustained delivery of NTFs also provides a potential for subsidiary nerve protection over short period (i.e., for months), which is regarded as key step before further medical treatments. A very effective means to improve functional axonal regeneration is by controlled delivery of multiple NTFs with their individually optimal kinetics [2,26]. To couple with the optimal kinetics for two or more proteins, suitable delivery systems must be developed to enable more efficient release kinetics. To overcome the long-standing delivery issues, gene transfer has emerged as a practical means of potentially providing the ‘enabling’ technology required for translating the use of these proteins into viable biotherapeutics for neurodegenerative diseases [11]. In addition, it is widely accepted that Schwann cells play an indispensable role in axonal regeneration. Therefore, efficient use of such glial cells at appropriate sites may improve regenerative efficacy, despite the fact that the isolation and culturing of persynaptic Schwann cells on large scale may present new challenges [38]. As a result of this, stem cells, like adipose-derived stem cells, have been proposed as alternative to obtain Schwann cells [39,40]. Although gene transfer and cells transplantation have shown mediate beneficial effects on nerve regeneration, clinically relevant protocols on safe and efficient use of them are yet to be developed.
4.3.
Unclear therapeutic window
The effect of NTFs are dose-dependent [11]. Currently, there is no available data concerning the therapeutic concentration of NTFs in targeted tissues or organs that can be used to evaluate the efficient therapeutic effect. To define the therapeutic window of NTFs, pertinent evaluation approaches should be
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developed. Ideally, the evaluation approaches should probably be practiced in scale and then the complicated biological effect would be assayed widely. Because of this, in vitro bioassay of NTFs is preferred, including the employment of PC12 cells and chicken embryonic dorsal root ganglia (DRG) methods. PC12 cells have been widely used as cell models for neuronal differentiation in response to exogenous signals, such as NTFs and neurotransmitters [41]. Previous reports demonstrated that effective NGF to stimulate PC12 cell neurite outgrowth is in the range of 0.1e50 ng/ml [42]. Another investigators applied DRG bioassay to optimize axonal growth response of NTFs and the results showed that the ideally dose was observed at 1e10 ng/ml of GDNF or NGF when used as single factors, whereas the optimal dose range was 0.1e1 ng/ ml for combined GDNF and NGF [9]. However, in vitro data should be ‘translated’ to in vivo information before clinical application, which met with enormous challenge. Some complications in a human clinical setting include genetic background, lifestyle, physical activity and age of the patients as well as variable pathology and additional medications, which all may affect the required dose. In conclusion, successful NTFs delivery requires dosage customization for each factor and delivery system, in both preclinical model and clinical case.
5.
Conclusion and summary
The reliable and meaningful clinical benefits of NTFs will intimately depend on their optimal localized delivery in a given context. A delivery system designed for nerve regeneration should (ⅰ) provide a time and dose-controlled release of the bioactive NTFs and (ⅱ) offer multiple NTFs delivery with individually optimal kinetics to achieve multi-functions (e.g. prevention of neuronal death and promotion of axonal growth). Additional requirements for a carrier include high biocompatibility, adequate biodegradability, ease of manufacturing (reproducibility and scale up), and cost effectiveness. Particular challenge for developing an optimal delivery systems for NTFs encompass the achievement of maintain optimal protein retention over a long period of time in target tissues. Optimizing and customizing sustained delivery systems of NTFs require continued attention but will hopefully lead to more efficient therapies for neurological disorders.
Acknowledgment This study was financially supported by the National Natural Science Foundation of China (Grant No. 81102401). We thank Xian Xu for editing the manuscript.
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