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Just Accepted to the Expert Opinion on Therapeutic Patents Estimated Publication date - 12 Feb 2015 (Online) , 28 Apr 2015 (Print) DOI: 10.1517/13543776.2015.1010512 © 2015 Informa UK, Ltd. ISSN 1354-3776, e-ISSN 1744-7674
This is an initial, pre-refereeing / pre-reviewing version. The final version, which was significantly improved after reviewers’ comments, will be available from: http://informahealthcare.com/loi/etp Authors are very thankful to Informa Healthcare and Expert Opinion on Therapeutic Patents for possibility to publish authors’ initial version. ––––––––––––––––––––––––––––––––––––––––––––––––––––Phosphorus-Containing Nanoparticles: Biomedical Patents Review Dzmitry Shcharbin 1, Natallia Shcharbina 2, Antos Shakhbazau 3, Serge Mignani 4, Jean-Pierre Majoral 5, Maria Bryszewska 6 * 1 – Institute of Biophysics and Cell Engineering of NASB, Minsk, Belarus, 2 – Republican Research and Practical Center of Neurology and Neurosurgery, Minsk, Belarus, 3 – Hotchkiss Brain Institute and Cumming School of Medicine, University of Calgary, Calgary, Canada, 4 – Université Paris Descartes, CNRS, Paris, France, 5 – Laboratorie de Chimie de Coordination, CNRS, Toulouse, France, 6 – Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Poland.
Corresponding author: *Prof. Maria Bryszewska, Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Poland.
Abstract: Introduction. The beginning of the nano-era started with the appearance of artificial nanosized supramolecular systems called nanomaterials. Their bursting on to the scene has been observed in both fundamental science and modern industry, and has led the appearance of new fields of R&D, notably nanotechnology, nanobiology, nanomedicine. Areas covered. In the present review, we have analyzed the patents on phosphorus-based nanomaterials (fullerenes, quantum dots, graphene, liposomes, dendrimers, gold and silver nanoparticles) in biology and medicine. Their impact in treatment of cancer, viral infections, and cardiovascular diseases is discussed. Expert opinion. Liposomes and dendrimers had the highest number of biomedical patents. The third candidates were quantum dots, and the fourth and fifth were gold and silver nanoparticles. Fullerenes and carbon nanotubes have the fewest applications in biology and medicine. Thus, our first conclusion was about the ‘unifying nanotoxicology paradigm’ [Kannan, Tomalia et al.], that ‘soft’ nanoparticles are significantly more biocompatible than ‘hard’ nanoparticles. The trend of applications of these nanomaterials is in medicine drug and gene delivery, visualization of cells and pathologic processes, using them as antivirals and antimicrobials, contrast agents, antioxidants and photosensitizers. It was unexpected that no patents were found in which phosphorus NPs were used in 3D printing of bones and other biological tissues. The conclusion reached is that nanomaterials are promising tools in future medical applications.
MAIN TEXT: 1. Overview of patent-based applications of phosphorus NP in biology and medicine. The beginning of the nano-era was with the appearance of artificial nanosized supramolecular systems called nanoparticles (NPs) [1-2] (Fig. 1). Their bursting on the scene has been observed both in fundamental science and modern industry and had led to their application in new fields of R&D, notably nanotechnology, nanobiology, nanomedicine. Analysis of publications in the field of NPs unveils an interesting feature ; although there are more publications on fullerenes, graphene, carbon nanotubes (CNTs), metal NPs, and quantum dots (QDs) than on dendrimers and liposomes, publications concerning the application of liposomes and dendrimers in biology and medicine predominate over other nanomaterials: liposomes in biology and medicine – 65% of publications, dendrimers – 39%, metal NPs – 29%, fullerenes – 13%, QDs – 11%, CNTs – 9%, graphene – >1%; analysis was conducted using SCOPUS instruments .
Fig. 1. Types of nanoparticles: 1 – liposomes, 2 – dendrimers, 3 – metal nanoparticles (including gold and silver NPs), 4 – quantum dots, 5 – carbon nanotubes, 6 –fullerenes, 7 – graphene (as 2Dnanoparticle) [1-2].
In this review, we will focus on the application of phosphorus (phosphorus-based) NPs in biology and medicine based on recent patents. Phosphorus is extremely important element in living organisms. On the one hand, it is a component of the body in being part of the composition of vital molecules such as DNA, RNA, proteins, phospholipids, etc. [4-5]. On the other hand, organophosphorus compounds can be dangerous in being nerve poisons, detergents, pesticides, etc. [4-5]. The application of phosphorus (phosphorus-based) compounds has been studied over last century, being very widely described [4-6]. Our aim has been to narrow this super-wide family to the phosphorus NPs as a new kind of phosphorus compound for modern nanobiology and nanomedicine, therefore the uses of phosphorus compounds as plant fertilizers etc. were excluded.
Using SciFinder® (accessed at 20-26 October 2014), 1298 patents were found by using the keywords 'phosphorus' (or 'phosphorus-based’) AND ‘nanoparticle’ (or ‘nanoparticles’); duplicates were excluded by SciFinder® tools (It should be noted that some free resources, e.g. http://www.freepatentsonline.com , have a higher number of patents. However, there are no tools to check duplicates or the same documents applied to different patent offices). The keywords ‘phosphorus NP’ AND ‘biological’ showed 275 patents, ‘phosphorus NP’ AND ‘biological’ AND NOT ‘plants’ – 227 patents, ‘phosphorus NP’ AND ‘pharmaceutical’ – 184 patents, ‘phosphorus NP’ AND ‘medical’ – 59 patents. The redistribution of patents on phosphorus NPs by their type in biology and medicine is shown in Table 1. Table 1. The redistribution of patents (number of patents) on phosphorus NPs by their type in biology and medicine (except plant biology). Analysed using SciFinder® tools. Phosphorus NP
Totally, applications 135 147 582 481 103 123 254 84 113
Fullerene QD CNT Graphene Dendrimer Liposome Metal NP Gold NP Silver NP
Biological (AND NOT plants) 15 33 31 16 40 88 28 19 23
9 27 15 11 34 106 21 15 15
3 6 15 8 11 13 11 2 7
Fig. 2. Percentage of patents on applications of phosphorus NPs in biology (but not plant biology). Analysed using SciFinder® tools.
The analysis of patents in the field of phosphorus NPs is close to that of publications on NPs (see above): patents concerning applications of liposomes and dendrimers in biology and medicine predominate over other nanomaterials: 72% and 39%, correspondingly (in biology). In contrast to NPs distribution by publications, however, the impact of gold NPs in biology (Fig. 2) is significantly higher – 23%. Then come QDs – 22%, silver NPs – 20%, fullerenes – 11%, CNT – 5% and graphene – 3%. From our analysis, we can incorporate the metal, gold and silver NPs because their patents overlap (unfortunately, the complex syntax queries induced errors in search engine). As our next step, we carried out a more detailed patent analysis of applications of phosphorus NPs in biology and medicine (Figs. 3-6).
150 114 98
23 19 21 14
Drug ..Drug carrier ..Drug delivery Gene ..Gene delivery DNA ..DNA delivery RNA ..RNA delivery Viral ..Antiviral Microbial ..Antimicrobial Protein ..Antibody ..Enzyme ..Vaccine Tussue… Contrast agent Oxygen… Photosensitizer Antioxidant *
Fig. 3. Phosphorus NPs and their applications by keywords (for example, ‘phosphorus NPs’ AND ‘drug’, ‘phosphorus NPs’ AND ‘viral’, etc. Analysis used SciFinder® tools). Antioxidant* = ‘antioxidant’ AND ‘biology’ because phosphorus NPs are also synthesized in presence of antioxidants. Tissue.. = tissue coatings, Oxygen.. = oxygen sensing.
There were 184 patents devoted to using phosphorus NPs as drugs (keywords 'phosphorus NPs' AND 'drug', 'phosphorus NPs' AND 'drug delivery', 'phosphorus NPs' AND 'drug carrier', duplicates were excluded). The next important application of phosphorus NPs concerned their ability to interact with proteins (eg conjugate with antibodies, conjugate/interact with enzymes, act a new kind of vaccines). The third main application of phosphorus NPs is their ability to bind and transfer genetic material (Fig. 3). Application of phosphorus NPs as antiviral agents has at least 39 patents (22 patents by keywords: ‘phosphorus NPs’ AND ‘antiviral’, 39 patents – by ‘phosphorus NPs’ AND ‘viral’), as antimicrobial agents – 32 patents. Phosphorus NPs can be used as contrast agents – 21 patents, antioxidants – 16 patents, tissue coatings – 14 patents, photosensitizers – 8 patents, oxygen sensing agents – 6 patents. A significant number of single patents were found in which additional benefits of phosphorus NPs for biology and medicine had been discussed.
We followed the above with an analysis of phosphorus NP-based patents by diseases (Fig. 4).
40 24 15
Fig. 4. Phosphorus NPs and their applications by disease (for example, ‘phosphorus NPs’ AND ‘Cancer’, Analysis used SciFinder® tools). Cardiovascu = cardiovascular diseases.
From the Fig. 4, it follows that most phosphorus NPs have been developed to treat cancer - 59 patents (tumour – 28 patents). Treatment of inflammation was the second target of phosphorus NPs – 24 patents. They are also used to treat viral infections (15 patents, including HIV and Ebola viruses, cardiovascular diseases – 11 patents, diabetes – 5 patents, amyloid-based and prion diseases (several patents). Other diseases have been mentioned in single patents. Continuing our overview on phosphorus NPs in biology and medicine, it was of interest to compare phosphorus NPs with regard to their main applications (Fig. 5).
Fig. 5. Comparison of phosphorus NPs regarding their applications in drug delivery (keyword ‘drug delivery’, cylinder) or cancer treatment (keyword ‘cancer’, rectangle). Analysis used SciFinder® tools.
Liposomes (phospholipid-based liposomes) are the most frequently used phosphorus NP for drug delivery (83 patents), such as in cancer treatment (61 patents). The second are phosphorus dendrimers – 23 and 11 patents, correspondingly, the third being phosphorus QDs – 16 and 17 patents, correspondingly. The main application of QDs in cancer treatment is visualization. It is important that in ~50% of patents, QDs are reported as being coated with polymer or dendritic surfaces to improve their biocompatibility. Other nanoparticles are much less used than these 3 classes, which is connected with the significantly higher biocompatibility of liposomes and dendrimers compared with other nanomaterials [7-9]. We finally checked patents in which all these nanoparticles were combined with one another to create multifunctional nanoconjugates, as presented in Table 2 and Fig. 6.
Table 2. Patents on phosphorus multifunctional nanoconjugates (NP 1 and NP 2) in all fields of science (e.g. keywords: ‘phosphorus’ AND ‘fullerene’ AND ‘dendrimer’). Analysis used SciFinder® tools. NP1
Fullerene Fullerene Fullerene Fullerene Fullerene Fullerene Fullerene Fullerene CNT CNT CNT CNT CNT CNT Dendrimer Dendrimer Dendrimer Dendrimer
QD CNT Graphene Dendrimer Liposome Metal NP Gold NP Silver NP Graphene Dendrimer Liposome Metal NP Gold NP Silver NP Liposome Metal NP Gold NP Silver NP
No. of patents (starting year) 4 (2007) 52 (2001) 27 (2010) 7 (2006) 2 (2007) 0 0 1 (2011) 117 (2008) 3 (2008) 2 (2004) 14 (2005) 6 (2010) 9 (2009) 7 (2007) 1 (2004) 0 1 (2006)
No. of patents (starting year)
QD QD QD QD QD QD QD
CNT Graphene Dendrimer Liposome Metal NP Gold NP Silver NP
9 8 4 9 4 7 4
Graphene Graphene Graphene Graphene Graphene
Dendrimer Liposome Metal NP Gold NP Silver NP
1 (2013) 0 19 (2011) 5 (2011) 4 (2011)
Liposome Liposome Liposome
Metal NP Gold NP Silver NP
2 (2011) 1 (2011) 2 (2011)
(2006) (2009) (2007) (2006) (2011) (2005) (2007)
It is noteworthy that (1) multifunctional nanoconjugates on the basis of liposomes, dendrimers and QDs had ~ 50-90% of the biomedical applications, while the same conjugates, on the basis of CNT, fullerene, graphene, metal, gold and silver NP, had 98%; growth of E. coli on a film of this nanosized graphene oxide was also inhibited. Treseder and Whiteside  used QD for the delivery of microbicides, fungicides, pesticides, therapeutic agents, biologics, diagnostic agents, dye and marker substances to specific locations in plants or animals. In some embodiments, the QDs may be substantially free of cadmium . Phosphorus NPs as antivirals Cui et al.  created lipophilic monophosphorylated derivatives of gemcitabine - a nucleoside analog in which the hydrogen atoms on the 2' carbon of deoxycytidine are replaced by fluorine - used both in chemotherapy and the treatment of viral infections. Gemcitabine NPs based on PLGA nanoparticles possess strong anti-tumour and anti-viral activities . Zhao  synthesized inorganic and organic NP with surface crosslinked non-polymeric organic amphiphiles called multivalent surface-crosslinked micelle particles. These inhibit viruses or bacteria from binding to a host cell, as used in the therapy for flu or AIDS . Phosphorus NPs as tissue coatings Hong and Tan  created bioactive glass nanofibers containing calcium phosphate nanoparticles, which have high mechanical strength and biological activity, such that they are used as advanced bone repairing and filling materials. Shukla and Craig  proposed NP-based adhesive composition to be used in dentistry, a dental adhesive composition consisting of a photocurable ionomer, radiopaque metal oxide nanoparticles, a phosphorus-containing acidic monomer, other polymerizable resin components, and a polar solvent that is used as a dental coating . Yang et al.  synthesized ferroferric oxide/calcium phosphate NPs for cell regulation and bone repair under the effect of a magnetic field. The ferroferric oxide NPs serve as the core and calcium phosphate serves as the shell, and thus can be directed by a magnetic field in cell regulation and bone repair . Zhang et al.  synthesized B-containing nanometer mesoporous bioactive glass based on inorganic and organic different salts and polymers. This bio-glass material has a macropore size of 300-500 μm and mesopore size of 5 nm, a spatial surface area of 265-194 m2/g, and can be used for drug or gene support, bone repair filling material, or 3-D scaffolds in bio-engineering.
Phosphorus NPs to treat pulmonary diseases Beck-Broichsitter et al.  developed biocompatible polymeric nanoparticles for the treatment of pulmonary hypertension or erectile dysfunction. They consist of a biocompatible polymer, a stabilizer and a drug; the product is nebulizable and a continuous release of the active agent can cover a period of up to 48 h . Gene transfection using phosphorus NPs Chiou et al.  proposed photothermal substrates for the selective transfection of cells. A special NP coated surface was developed that heats up when contacted by electromagnetic radiation. The pores in this surface are in fluid communication with microchannels providing the reagents that can be delivered into the cells. Gold nanoparticles are synthesized directly on glass microcapillaries. Highly localized transient openings of the cell membrane are generated by the photothermal effect of nanoparticles on glass micropipets, with the dimension of a typical opening close to the micropipet tip size of ~2 μm. The cell remains viable after the procedure . Our team had also earlier reported the use of phosphorus-based dendrimers for neurotrophin gene transfection into human stem cells [36-37]. 3. Other interesting patents in biology connected with phosphorus compounds and NPs Briefly it is worth reporting that NPs can also be applied in biology as new generation fertilizers or to detect organic phosphorus compounds. Three interesting examples are given. NP for detection of phosphorus poisons As mentioned above, organic phosphorus compounds can be poisons for humans and animals. We found several patents in which NPs have been used to detect organic phosphorus compounds. In a typical patent, Wang et al.  proposed an NP-based enzyme sensor for detecting organic phosphorus pesticides. In this NP, acetylcholinesterase and Au-SiO2 nanoparticles are layered on a glassy carbon electrode. The enzyme sensor has the advantages of wide linear detection range (6.0 × 10-13-2.0 × 10-1 mol/L) and low detection limit (3.0 × 10-13 mol/L) . Biosynthesis of metal NPs from fungi Tarafdar and Raliya  provided a low cost, eco-friendly and rapidly synthesised agriculturally important metal NP containing phosphorus, magnesium, zinc, iron and titanium. It is based on the incubation of cell-free filtrate of extracellular fungal secretions with aqueous solutions of the salts of these compounds. Phosphorus NPs as agricultural nutrients In plant biology, phosphorus NPs are extensively studied as carriers of nutrients or fertilizers. Kottegoda et al.  proposed a fertilizer in which a macronutrient (urea) is adsorbed on the surface of hydroxyapatite phosphate nanoparticles. Such a fertilizer slowly releases the macronutrient into the soil. 4. Conclusions We need to ask first: what do biology and medicine expect from nano-based materials? Initially it means overcoming existing barriers of current drugs by means of (1) longest circulation and action, (2) enhanced defense against premature biodegradation, (3) increased bioavailability, (4) overcoming of all body barriers, (5) targeted and controlled release, (6) minimal adverse effects, and (7) complexicity of action to minimize application (multifunctional nanoconjugates might provide simultaneous diagnosis and targeted treatment of pathologic process). As seen from the patents, phosphorus NPs seem to resolve all these problems because they can successfully deliver drugs and genetic material, be effective drugs in themselves, act as anti-virals and anti-microbials, operate as new kinds of tissue coatings and contrast agents, amongst other things. Thus, their development is prospective way to create new biomedical nanomaterials.
5. Expert opinion The explosive development in nanomaterials has led to extensive application of nanoscale materials in biological processes and attempts to create new nano-based tools for traditional medicine. This has led to the appearance of the new disciplines of nanobiology and nanomedicine. The unique features of nanomaterials allow them to preferentially penetrate and be retained by biological cells and tissue, and there have been developments of ways to detect and image nanomaterials in cells and biological tissue. Moreover, as constructor details, they can be combined into multi-functional nanodevices and nanoconjugates. Such conjugates will allow multiplexing, ie, providing several functions in one conjugate (e.g. drug carrier plus probe for visualization). However, we must also be aware that different kinds of nanomaterials can have drastically different behaviour in biological tissue. In this regard it is interesting to point out their differences based on patent analysis. A. The most promising phosphorus NPs for medicine Liposomes and dendrimers show the greatest number of biomedical patents, the third candidates being QDs, and the fourth and fifth being gold and silver NPs (both metal NP). Fullerenes and CNTs have the fewest applications in biology and medicine, since they are insoluble in most organic or aqueous solutions, and surface modification is critical for their use in medicine . Thus, the data confirm the suggestion that there is a significantly higher biocompatibility of liposomes and dendrimers compared with other kinds of nanomaterials [7-9]. Gebel et al. , after extensive analysis of nanomaterials, made some important conclusions: first, there is currently no evidence of ‘nanospecific’ mechanisms of action; and no step-change in hazard has been observed so far for particles